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Articles by Mahesh P. Burse in JoVE

 JoVE Applied Physics

Bringing the Visible Universe into Focus with Robo-AO

1Caltech Optical Observatories, California Institute of Technology, 2Department of Astronomy, California Institute of Technology, 3Dunlap Institute for Astronomy and Astrophysics, University of Toronto, 4Inter-University Centre for Astronomy & Astrophysics, 5Observatories of the Carnegie Institution for Science, 6Benoziyo Center for Astrophysics, Weizmann Institute of Science


JoVE 50021

Light from astronomical objects must travel through the earth's turbulent atmosphere before it can be imaged by ground-based telescopes. To enable direct imaging at maximum theoretical angular resolution, advanced techniques such as those employed by the Robo-AO adaptive-optics system must be used.

Other articles by Mahesh P. Burse on PubMed

Chlorinated Hydrocarbon Pesticides in the Fat of People in New Orleans

A Reciprocal Relationship Between the Induction of -aminolevulinic Acid Synthetase and Drug Metabolism Produced by M-dichlorobenzene

Comparison of the in Vitro and in Vivo Tarnish of Three Gold Alloys

Chlorinated Hydrocarbon Pesticides and Related Compounds in Adipose Tissue from People of Japan

Polychlorinated Biphenyls: Evidence of Transplacental Passage in the Sherman Rat

Adenofibrosis in the Rat Liver, with Persistence of Polychlorinated Biphenyls in Adipose Tissue

Chlorodibenzo-p-dioxin Contamination of Two Commercially Available Pentachlorophenols

Long-term Occupational Exposure to DDT

Polychlorinated Biphenyls. Storage, Distribution, Excretion, and Recovery: Liver Morphology After Prolonged Dietary Ingestion

Evidence of Chlorodibenzo-p-dioxin and Chlorodibenzofuran in Hexachlorobenzene

Induction of Liver Tumor in Sherman Strain Female Rats by Polychlorinated Biphenyl Aroclor 1260

Sherman strain female rats (200) were fed 100 ppm of polychlorinated biphenyl (Aroclor 1260) for apporximately 21 months, and 200 female rats were kept as controls. The rats were killed when 23 months old. Twenty-six of 184 experimental animals and 1 of 173 controls had hepatocellular carcinomas. None of the controls but 146 of 184 experimental rats had neoplastic nodules in their livers, and areas of hepatocellular alteration were noted in 28 of 173 controls and 182 of 184 experimental animals. Thus the polychlorinated biphenyl Aroclor 1260, when fed in the diet, had a hepatocarcinogenic effect in these rats. The incidence of tumors in other organs did not differ appreciably between the experimental and control groups.

[Occlusion: Notes of Common Interest of Dental Specialties]

A Comparative Study of Two Polychlorinated Biphenyl Mixtures (Aroclors 1242 and 1016) Containing 42% Chlorine on Induction of Hepatic Porphyria and Drug Metabolizing Enzymes

Evidence of Tetrachlorodibenzofuran (TCDF) in Aroclor 1254r, and the Urine of Rats Following Dietary Exposure to Aroclor 1254

Comparison of Physiological Responses of Women and Men to Isometric Exercise

The volunteers for this study were 83 women, aged 19-65 yr, drawn from several different occupations. Three minutes after exerting maximal handgrip strength (MVC) each subject held a tension of 40 percent MVC to fatigue. Blood pressures and heart rates were measured before, during, and after the endurance of contraction. Age was associated with a reduction of strength of the women, whereas their endurance at 40 percent at 40 per cent MVC increased. There was evidence that menopause enhanced those age effects for strength and endurance. At rest, age was associated with a decreased heart rate. As expected, the heart rates of all the women increased during the endurance contraction. But that increase was greater for the younger than for the older women, thereby exaggerating the difference due to age already seen at rest. Systolic blood pressure at rest was higher with age and, in a similar manner, that relationship was also exaggerated throughout the isometric contraction. Diastolic blood pressure, however, was not changed with age at rest, and although the diastolic pressure increased during the isometric exercise, the rate of increase was unaffected by age. The results obtained are compared with those from a similarly large number of men examined in identical circumstances.

The Assessment of the Static Component in Rhythmic Exercise

A new approach has been devised to assess the "static component" of dynamic exercise. This technique involves the measurement of the isometric endurance of muscles which have just taken part in rhythmic exercise and depends on the repeatability of trained subjects in isometric effort. The premise is that isometric endurance will be inversely related to the static component of the preceeding dynamic exercise. The subjects worked on a bicycle ergometer at known fractions of their maximal aerobic capacity (max Vo2). The rate of pedalling was varied from 30 to 90 rpm, so that for a given % max Vo2, the belt tension varied inversely with the speed of cycling. At any one speed of cycling, isometric endurance decreased as the belt tension increased. Following exercise at 30 rpm, the isometric endurance was 25 to 50% lower than that found at the most advantageous speed of cycling for our subuects; at these faster rates of cycling two subjects showed least static component following exercise at 90 rpm while the remaining subject performed best after cycling at 50 rpm.

Differentiated Ratings of Perceived Exertion During Physical Conditioning of Older Individuals Using Leg-weight Loading

Use of leg weights for physical conditioning was evaluated in 8 middle-aged male Ss; four Ss of similar age served as a control group. Pre- and post-training evaluation consisted of heart rate and oxygen uptake responses to five submaximal work loads which involved either level walking or cycling. Differentiated ratings of perceived exertion elicited for each work load were: a local muscular rating; a central or cardio-pulmonary rating; and an over-all or general rating. Submaximal heart rate decreased 6 to 9 beats/min. from pretraining values for all work load after training. The differentiated ratings for training generally reflected a reduced strain on the cardiovascular system and also improved functioning of the working muscles with training. However, when one set of sensations dominated the exertional perception the others appear to have been perceptually de-emphasized. Local muscular factors seemed to dominate the exertional perception for cycling, but central factors appeared to play a more important role for treadmill walking, at least within the range of velocities investigated.

A Comparison of Analytical Methods for Chlorodibenzo-p-dioxins in Pentachlorophenol

PCB Metabolism in Rats Following Prolonged Exposure to Aroclor 1242 and Aroclor 1016

Several mono- and dihydroxy metabolites of di-, tri, and tetrachlorobiphenyl have been identified in the urine of rats fed prolonged diets of Aroclor 1016 or Aroclor 1242. Combined gas chromatography-mass spectrometry was used for characterization of the metabolic products.

Toxicity of Polybrominated Biphenyl

[Preparation of the Patient and His Mouth for Receiving a Partial Removable Denture]

Role of Physical Fitness in Heat Acclimatisation, Decay and Reinduction

Epidemic Kepone Poisoning in Chemical Workers

From March 1974 through July 1975, 76 (56%) of 133 persons who had worked at a pesticide plant that produced Kepone, a chlorinated hydrocarbon insecticide, contracted a previously unrecognized clinical illness characterized by nervousness, tremor, weight loss, opsoclonus, pleuritic and joint pain, and oligospermia. Illness incidence rates for production workers (64%) were significantly higher than for nonproduction personnel (16%). The mean blood Kepone level for workers with illness was 2.53 ppm and for those without disease 0.60 ppm (p less than 0.001). Blood Kepone levels in current workers (mean, 3.12 ppm) were higher than those in former employees (1.22 ppm). Blood Kepone levels for workers in nearby businesses and for residents of a community within 1.6 km of the plant ranged from undetectable to 32.5 ppb. Illness attributable to Kepone was found in two wives of Kepone workers; there was no apparent association between frequency of symptoms and proximity to the plant in the survey of the community population.

Persistent Liver Lesions in Rats After a Single Oral Dose of Polybrominated Biphenyls (firemaster FF-1) and Concomitant PBB Tissue Levels

In a preliminary study, 12 male and 12 female weanling Sherman strain rats were given a single dose of 1000 mg polybrominated biphenyls (PBBs) FireMaster FF1 Lot 7042 kg/body weight as a 5% solution in corn oil. Three male and three female weanling rats were given corn oil. One day after dosing PBB blood levels ranged from 78 to 162 ppm and 42 days later they ranged from 1.1 to 2.99 ppm. The liver was the only organ with pathological changes. In a long-term recovery study groups of 20 male and female rats, 2 months old, were given 0 or 1000 mg PBBs/kg body weight as a single dose in peanut oil. Five rats per group killed 2, 6, 10, and 14 months after dosing had pronounced liver pathology, including hepatic porphyria in the female rats and neoplastic nodules also mainly in female rats. Chemical analyses of blood, liver, and adipose tissue for PPBs 10 and 14 months after dosing gave the following mean results. Blood levels in females were 2.9 and 2.92 ppm, respectively, and males 0.94 and 1.34 ppm, respectively. Adipose tissue levels in females were 1202 and 783 ppm and in males 713 and 866 ppm, respectively. The liver levels in females were 37 and 22 ppm and in males 60 and 63 ppm, respectively.

Influence of Posture on Isometric Fatigue

The isometric strength of four trained subjects was unaltered by changes in posture. But the endurance of an isometric contraction held to fatigue at 25 and 40% of the maximum voluntary contraction (MVC) was 20% greater in the sitting than in the recumbent posture. This difference was abolished when the exercise was performed with the arm's circulation arrested. At rest, the blood flow through the forearm was greater when the subjects were in the recumbent than in the sitting position but the reverse was true during isometric contractions. In these two postures, there was no difference in the right atrial pressure during the contraction, suggesting that the low-pressure baroreceptors are not responsible for the differences in blood flow during exercise. To date no mechanism is available to explain these observations.

Sex Differences in Human Thermoregulatory Response to Heat and Cold Stress

Physical Conditioning of Sedentary Young Men with Ankle Weights During Working Hours

The Effect of Diet on Thermogenesis in Acquired Lipodystrophy

Interlaboratory Comparison for Results of Analyses for Polybrominated Biphenyls in Human Serum

Four hundred and sixty-six "blind" duplicate serum samples were analyzed for polybrominated biphenyls (PBB) by gas chromatography in two laboratories. As calculated by the regression model of Deming, the correlation coefficient for these 466 samples, whose values ranged from nondetectable amount to 1240 parts per billion was 0.9983. Prior to these analyses, the method was validated in both laboratories by using in vitro and in vivo PBB serum pools.

The Effect of Different Diets or Mineral Oil on Liver Pathology and Polybrominated Biphenyl Concentration in Tissues

Metabolic Consequences of Exposure to Polychlorinated Biphenyls (PCB) in Sewage Sludge

Polychlorinated biphenyls (PCB) were discovered in sewage sludge used for fertilizer in Bloomington, Indiana. The PCB had been discharged into the municipal sewage system by an electrical capacitor manufacturing plant. To study the epidemiology and metabolic consequences of PCB exposure an epidemiologic and clinical survey was conducted. Mean serum PCB levels were 17.4 ppb in 89 sludge users, 75.1 ppb in 18 workers with occupational exposure to PCB, 33.6 ppb in 19 members of those workers' families, and 24.4 ppb in 22 community residents without unusual exposure to PCB. In sludge users PCB levels were associated positively with per cent performance of garden care (p = 0.035) and negatively with wearing gloves while gardening (p = 0.021), but were not significantly associated with the amount of sludge used or the duration of exposure. In no groups were chloracne or systemic symptoms of PCB toxicity noted, nor were significant correlations found between PCB levels and tests of hematologic, hepatic, or renal function. Plasma triglyceride levels increased significantly with serum PCB concentrations in both alcohol drinkers and nondrinkers (r = 0.541, n = 36, p < 0.001 for nondrinkers). These data indicate that PCB may alter lipid metabolism at levels of exposure and bioaccumulation insufficient to produce overt symptoms.

Induction of Liver Tumors in Female Sherman Strain Rats by Polybrominated Biphenyls

Noninbred Sherman strain rats were given the polybrominated biphenyl mixture Firemaster FF-1 (PBB). Rats given a single dose of 1,000 mg PBB/kg or 12 doses of 100 mg PBB/kg body weight in corn oil by gavage had final (when less than or equal to 26 mo old) liver PBB concentrations of 17.1 and 34.8 mg/kg (wet wt), respectively. The respective incidence of hepatocellular carcinomas was 41.4 and 67.8%. No difference in PBB concentrations was found between hepatocellular carcinomas and surrounding liver tissue. In addition, most livers of PBB-dosed rats had adenofibrosis of the liver. Livers of controls were essentially normal. Rats given a single dose of 200 mg PBB/kg as above had a 31.2% incidence of neoplastic nodules, whereas none were seen in the controls. The mean PBB concentrations (when 26 mo old) were 2.68 mg/kg in liver, 244 mg/kg in adipose tissue, and 0.22 mg/kg in blood.

Temperature-programmed Gas Chromatographic Determination of Polychlorinated and Polybrominated Biphenyls in Serum

An analytical method was developed to quantitate polychlorinated and polybrominated biphenyls (PCBs and PBBs, respectively) in human serum. The method includes denaturation of the proteins in serum, extraction, adsorption chromatography, and gas chromatography with electron capture detection. The coefficients of variation for determining the in vivo bound PCBs and PBBs ranged from 11.7 to 29.8% and 7.1 to 14.0%, respectively. The method is capable of measuring 10 ng PCBs and PBBs/mL in 4 mL serum.

The Effect of Deep Muscle Temperature on the Cardiovascular Responses of Man to Static Effort

Eight healthy male subjects (age range 24-38 year) were asked to exert a fatiguing isometric endurance contraction with their handgrip muscles at 40% of their maximum strength after immersion of their forearms in water at various temperatures ranging from 3-40 degrees C. For each subject, isometric endurance was longest after immersion of his forearm in water at a particular characteristic bath temperature; endurance decreased markedly above or below this temperature. The increase in heart rate from the beginning to the end of the fatiguing contractions was the same irrespective of the bath temperature. In contrast, the increase in blood pressure (both systolic and diastolic) throughout the contractions was almost constant for contractions exerted after immersion of the forearm in water at 20-40 degrees C, but was reduced progressively for contractions exerted in water below 20 degrees C.

Phenytoin: Ineffective Against Acute Mountain Sickness

Phenytoin sodium was evaluated for its effect on the development and intensity of acute mountain sickness (AMS) because of its ability to reduce intracellular Na+ concentrations in brain and thereby minimize any tendency to increase cellular volume, a hypothetical cause of AMS. Six men aged 19-35 were exposed to approximately 4600 m altitude in a hypobaric chamber for 52 h on two occasions separated by 10 d at sea level. Subjects received wither phenytoin or placebo for 18 h before (700 mg, divided dose) and throughout (100 mg t.i.d.) each altitude exposure in a double-blind, repeated-measures (crossover) design. Phenytoin serum concentrations ranged from 4.4-13.9 micrograms/ml during altitude exposure. Twice daily questionnaires and clinical evaluations showed no marked benefit from phenytoin on the occurrence, severity, or duration of AMS symptoms: headache, nausea, insomnia, and general malaise. Overall, 1 subject felt better, 2 felt worse, 1 felt the same; 2 were not suitably comparable. There was no observed relationship between serum levels and symptoms of AMS. Moderate degrees of weakness and dizziness were each reported by 3 subjects with phenytoin but not with placebo, however. Resting pulmonary ventilation, end-tidal PO2 and PCO2, map reading abilities and respiratory mask donning times were not affected by phenytoin. Under the conditions of this trial, phenytoin did not appear to be useful in managing AMS.

Isometric Fatigue Induced by Different Levels of Rhythmic Exercise

Three subjects were trained in leg extensor isometric contractions and in cycling. They then cycled for three consecutive bouts, each of 2.75 min at a constant level of VO2, from 20 to 80% VO2 max. Fifteen seconds after each bout of cycling the subjects exerted an isometric contraction of the right leg at 40% of the maximum voluntary contraction. In each experiment, the duration of the three successive isometric contractions decreased as in hand-grip contractions. There was also a linear reduction in isometric endurance as the severity of the preceding rhythmic exercise increased. In other experiments, after three bouts of rhythmic exercise at 20% VO2 max (each followed by a fatiguing contraction at 40% MVC), further bouts of cycling at increasing levels of severity up to 60% VO2 max resulted in a linear fall in isometric endurance which could be reversed by interposing a lighter level of cycling. The heart rates during these experiments showed a steady increase during the isometric exercise, to about 150 beats X min-1, as the bouts of preceding rhythmic exercise became progressively more severe. The isometric contractions had little influence on the heart rate during cycling. But the rhythmic cycling exercise markedly increased the heart rate achieved at the end of the isometric contractions while decreasing the increment in heart rate during the contraction.

Design and Demonstration of a Quality Assurance Program for Labor-intensive Analytical Systems

An interlaboratory quality assurance program was designed and implemented for the analysis of serum for polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). The two primary means of quality control were analysis of known and blind quality control samples and analysis of blind duplicate serum samples.

Gas-liquid Chromatographic Determination of Polychlorinated Biphenyls and a Selected Number of Chlorinated Hydrocarbons in Serum

A method is proposed for concurrently determining the levels of multiple intact exogenous compounds in serum, particularly polychlorinated biphenyls (PCBs) as Aroclor (AR) 1254 and chlorinated hydrocarbons (CHs). Bovine serum pools containing in vivo-bound PCBs (as AR 1254) and in vitro-spiked CHs are used to evaluate the method, which encompasses serum denaturation with methanol, mixed solvent extraction, multiple solvent fractionation from activated silica gel, and determination by electron capture gas-liquid chromatography. Mean recoveries of the in vitro-spiked 9 CHs at levels of 2.0-29.1 ppb ranged from 52.8 to 98.4% from trial environmental pools; mean recoveries of the in vivo-bound PCBs (as AR 1254) were 114.1 and 92.6% at levels of 10 and 50 ppb, respectively.

Assessment of Methods to Determine PCB Levels in Blood Serum: Interlaboratory Study

Twenty-five laboratories participated in a study to determine the levels of polychlorinated biphenyls (PCBs) in 3 bovine serum pools, referred to as trial environmental pools (TEPs). TEPs 2 and 3 contained, respectively, low (9.97 ppb) and high (49.64 ppb) levels of PCBs (as in vivo-bound Aroclor 1254) and the same level of 9 commonly occurring chlorinated hydrocarbons. TEP 1 contained only naturally occurring levels of these analytes. Laboratories analyzed each sample in duplicate by the method used in their laboratory for measuring PCBs in blood serum. The coefficients of variation (CV) for the 12 laboratories reporting quantitative data and the required number of analyses for TEP 2 and TEP 3 were 37.0 and 30.7%, respectively. The mean recoveries for these 12 laboratories were 239.3 and 165.4% for TEP 2 and TEP 3, respectively. Three laboratories reported data with mean values for TEP 2 and TEP 3 within +/- 3 standard deviations of the CDC characterized mean. Their coefficients of variation were 12.4 and 18.8% for TEP 2 and TEP 3, respectively. The mean recoveries for these 3 laboratories were 150.7 and 98.4% for TEP 2 and TEP 3, respectively. Our most significant observations were the laboratories' failure to separate PCBs from DDTs and the excessive background of the reagent blanks. The widely discrepant results indicate a definite need to standardize methodology for this analysis.

Evaluation of Potential Analytical Approach for Determination of Polychlorinated Biphenyls in Serum: Interlaboratory Study

Forty-four laboratories participated in evaluation of a method for determining polychlorinated biphenyls (PCBs) as AR 1254 in serum at the parts per billion level. The method involves deproteinating serum with methanol, extracting with hexane-ethyl ether, and eluting PCBs from deactivated silica gel for gas-liquid chromatographic determination with electron capture detection. Compounds are quantitated by using the Webb-McCall factors. Five serum pools, 4 containing in vivo-fortified PCBs (as AR 1254) or 8 in vitro-fortified chlorinated hydrocarbons (CHs), or both, were used. For PCB fortification levels of 9.89 (EP 2), 24.74 (EP 3), and 74.20 ppb (EP 4), interlaboratory coefficients of variation (CVs) for collaborators that adhered to protocol were 92.7, 67.6, and 25.8%, respectively. CVs on the same pools analyzed by the Centers for Disease Control (CDC) were 7.4, 7.8, and 4.6%, respectively. Average interlaboratory recoveries for pools EP 2, EP 3, and EP 4 were 138.1, 111.2, and 91.1%, respectively, and 99.8, 89.6, and 90.4%, respectively, for CDC on the same pools. There was a general decrease in the mean error for those laboratories that had participated in an earlier study in which they were allowed to use their own methods.

Procedures for the Measurement of Acute Mountain Sickness

Although acute mountain sickness (AMS) has been studied for well over a century, a standard measure or index of the degree of illness for use in experimental research does not exist. This paper outlines a definition and procedures for an operational measurement of AMS using the Environmental Symptoms Questionnaire (ESQ). After 58 men completed over 650 ESQs during a stay of 1-3 weeks atop Pike's Peak (4300 m), factor analysis produced nine distinct symptom groups, with two factors representing AMS. The first factor contains symptoms indicative of cerebral hypoxia and is labeled AMS-C. The second reflects respiratory distress and is called AMS-R. Signal detection theory was used to establish a criterion score value for each factor. Standard deviation values were used to derive indices of sickness severity. Discussion is given to the possible relationships between the two types of AMS and the more serious conditions of cerebral and pulmonary edema.

Height, Weight, Percent Body Fat, and Indices of Adiposity for Young Men and Women Entering the U.S. Army

The purpose of this investigation was to describe the height (H), weight (W), and percent body fat (%BF) of young men and women (ages 17-35 years) entering the U.S. Army and to determine an index of adiposity that fit criteria described in the literature. H and W were measured with a digital scale and anthropometer, respectively. %BF was calculated from four skinfolds thickness. Men and women were both separated into four age categories. Very little difference in H was found with increasing age. W and %BF increased progressively with age in the males but no increase in either parameter was seen within the three youngest age groups of women. For males, W/H2 was found to be the most appropriate index of adiposity of those studied, having a correlation with %BF of 0.75 and a standard error of estimate of +/- 3.4 %BF. W/H1.5 was the most appropriate index for females, having a correlation with %BF of 0.69 and a standard error of estimate of +/- 3.2 %BF. It was suggested that these indices could be used to replace or supplement the current H-W charts used in the Army. A table for predicting %BF from these indices has been provided.

The Influence of Cardiorespiratory Fitness on the Decrement in Maximal Aerobic Power at High Altitude

There are conflicting reports in the literature which imply that the decrement in maximal aerobic power experienced by a sea-level (SL) resident sojourning at high altitude (HA) is either smaller or larger for the more aerobically "fit" person. In the present study, data collected during several investigations conducted at an altitude of 4300 m were analyzed to determine if the level of aerobic fitness influenced the decrement in maximal oxygen uptake (VO2max) at HA. The VO2max of 51 male SL residents was measured at an altitude of 50 m and again at 4300 m. The subjects' ages, heights, and weights (mean +/- SE) were 22 +/- 1 yr, 177 +/- 7 cm and 78 +/- 2 kg, respectively. The subjects' VO2max ranged from 36 to 60 ml X kg -1 X min -1 (mean +/- SE = 48 +/- 1) and the individual values were normally distributed within this range. Likewise, the decrement in VO2max at HA was normally distributed from 3 ml X kg-1 X min-1 (9% VO2max at SL) to 29 ml X kg-1 X min-1 (54% VO2max at SL), and averaged 13 +/- 1 ml X kg-1 X min-1 (27 +/- 1% VO2max at SL). The linear correlation coefficient between aerobic fitness and the magnitude of the decrement in VO2max at HA expressed in absolute terms was r = 0.56, or expressed as % VO2max at SL was r = 0.30; both were statistically significant (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Polychlorinated Biphenyls and Lipemia on Serum Analytes

Twelve serum analytes [triglycerides, cholesterol, total and conjugated bilirubin, high-density-lipoprotein cholesterol (HDL-C), alkaline phosphatase (AP), gammaglutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), beta-glucuronidase (beta-glu), alanine aminopeptidase (AAP), and 5'-nucleotidase (5'nuc)] were measured to investigate their correlation with exposure to polychlorinated biphenyls (PCBs) and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The relationship between serum lipids, lipophilic toxicants, and the analytes was also evaluated. The beta-glu, 5'nuc, triglycerides, cholesterol, and total bilirubin correlated positively and significantly with log concentrations of serum total PCBs and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT. The more highly chlorinated PCBs (Aroclor 1260) had significant, positive correlations with several serum analytes, but the less chlorinated PCBs (Aroclor 1242) correlated significantly and negatively only with HDL-cholesterol. Triglyceride- and cholesterol-rich lipoproteins were added to serum to determine the effects of lipids on these assays. Several were spuriously elevated. AP and beta-glu were not affected by lipoprotein addition with the methods used in this study. AAP was increased significantly only at triglyceride concentrations exceeding 400 mg/dl. Lipoproteins may be elevated because of deranged lipid metabolism in response to PCBs, or PCBs may be elevated because elevated lipoproteins are present, as in familial triglyceridemia, a relatively common dyslipoproteinemia. Because this relationship is not well understood with respect to cause and effect, we propose the further use in epidemiological investigations of assay methods that are little affected by blood lipids yet are correlated with PCB concentrations. Congener-specific quantification of PCBs would help elucidate the effects of PCBs on assays used to monitor health effects.

Evaluation of Persons Exposed to Dairy Products Contaminated with Heptachlor

Convulsions Caused by Endrin Poisoning in Pakistan

From July through September 1984, acute convulsions caused by endrin poisoning occurred in the subdistrict of Talagang, Attock District, Punjab province, Pakistan. Eighteen of the 21 affected villages were surveyed; 70% of the cases for which ages were known (106 of 152) were in children 1 to 9 years of age; 9.8% of all affected persons (19 of 194) died. The outbreak occurred in villages on the main roads of the subdistrict and peaked in early September. Endrin was detected in the blood of 12 of 18 patients with a history of convulsions but was not found in the blood of four hospitalized control patients. One composite sugar sample taken from the homes of three persons had an endrin level of 0.04 ppm. Because of the high toxicity, repeated association with large-scale outbreaks of neurologic illness, and the difficulties of monitoring distribution, endrin should not be used for agricultural purposes.

Effect of Dexamethasone on Symptoms of Acute Mountain Sickness at Pikes Peak, Colorado (4,300 M)

In a previous controlled study, dexamethasone (DEX) was shown to prevent acute mountain sickness (AMS) during exposure to simulated high altitude. To determine the effect of DEX during actual altitude exposure, 16 young men were treated with either DEX (4 mg every 6 h) or placebo for 48 h prior to and 48 h after being rapidly transported from sea level to the summit of Pikes Peak, CO (4,300 m). Symptoms of AMS were evaluated twice daily at Pikes Peak using the Environmental Symptoms Questionnaire and a clinical assessment. During treatment the mean symptom scores were higher for subjects taking placebo in 18 out of 20 comparisons. On an individual basis, 60% of the subjects receiving placebo met the criteria for being "sick" compared to 31% of subjects receiving DEX. Beginning 24 h after cessation of treatment, DEX subjects experienced a progressive increase in symptom scores which lasted through the end of the altitude sojourn (day 6). The results indicate that DEX is an effective prophylactic treatment for AMS in an actual mountain environment, but that AMS symptoms can occur if the drug is stopped abruptly.

Organochlorine Recovery from Small Adipose Samples with the Universal Trace Residue Extractor (Unitrex)

Respiratory Response and Muscle Function During Isometric Handgrip Exercise at High Altitude

The purpose of this investigation was to determine if the hyperventilatory response to fatiguing isometric exercise at sea level could predict resting ventilation and acute mountain sickness (AMS) at 4300 m altitude. Exercise consisted of four successive endurance handgrips held to complete fatigue at 40% of maximum isometric handgrip strength (MHS). There was no relationship between the magnitude or pattern of exercise-induced hyperventilation at sea level and the severity of AMS later at altitude. Sea level hyperventilatory response was not predictive of resting ventilation at altitude. Altitude exposure progressively increased both the incidence and magnitude of the hyperventilatory response to exercise and prolonged it for 60-90 s into the recovery period, providing support for the "central command" theory of ventilatory control during isometric exercise. MHS was significantly increased at altitude--by 11% on day 2 and 16% on day 6. Endurance times to fatigue were reduced, but not always significantly so. A follow-up study involving more practice at sea level demonstrated MHS to be significantly increased throughout an entire 18-d stay at 4300 m and for 3, but not 5, days after descent. Significant changes in endurance could not be demonstrated. Neither AMS nor changes in body weight or circulating norepinephrine levels can account for the temporal pattern of increased grip strength, but the respiratory alkalosis occurring at altitude appears to be a likely mechanism.

Persistence of Heptachlor in Serum of People Consuming Contaminated Dairy Products

Acute Mountain Sickness at 4500 M is Not Altered by Repeated Eight-hour Exposures to 3200-3550 M Normobaric Hypoxic Equivalent

A lightweight device, designed to supply inspired air at 12.8% O2 concentration (PO2 equivalent to 3960 m altitude) by recirculating a portion of each expired breath after CO2 removal, was tested at sea-level for its ability to induce altitude acclimation. Twelve young men (experimental group) breathed from the device for 7.5-8 h each day for 10 successive days. On the morning of day 1, inspired O2 concentrations averaged 12.8%, as intended, but increased by noontime and remained elevated thereafter. This raised the average hypoxic stimulus to 13.8 +/- 0.9% (PO2 equivalent to 3370 +/- 517 m altitude) for the entire 10-d period. Ten other young men (control group) breathed normoxic air from a placebo device of identical appearance on the same schedule. On day 10, both groups were exposed for the next 2 d to 4500 m altitude in a hypobaric chamber to assess the effect of the treatment on acute mountain sickness (AMS). After the sea level treatment, the experimental group showed no significant differences from control in resting ventilatory rate, respiratory frequency or end tidal PO2, but end-tidal PCO2 was lower; there was no indication of hemoconcentration. At altitude, both groups showed the expected decreases in end-tidal PO2 and PCO2, and increases in hemoglobin concentration and hematocrit indicative of hemoconcentration, with no differences between them. Neither incidence nor severity of AMS differed significantly between groups, but the experimental group had a lower incidence rate than historical controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Exposure to Polychlorinated Biphenyls at Toxic Waste Sites: Investigations in the United States

Beginning in 1982, environmental and population data were evaluated from waste sites contaminated with polychlorinated biphenyls (PCBs). Pilot exposure assessment studies were conducted at 12 sites where risks of human exposure were thought to be greatest. Serum PCB levels in persons at highest risk of nonoccupationally related exposures (because of their self-reported frequencies and types of activities in contaminated areas) at 10 sites were within background ranges, even though environmental contamination levels as high as 2.5 parts per billion (ppb) in monitoring well water samples and 330,000 ppb in soil samples were measured. At the 2 remaining sites, elevated serum levels were found in these high-risk persons, which require further evaluation by community surveys. These results illustrate that, despite elevated environmental contaminant levels, unless uptake of chemicals above background exposure levels can be demonstrated, adverse health effects cannot be attributed to waste site chemicals. However, health risks due to background exposure levels, as well as in populations with elevated PCB body burdens need further study.

Comparison of Two Techniques for Quantifying Environmental Contaminants in Human Serum

Peak area matching and linear regression were used to quantify eight chlorinated pesticides and polychlorinated biphenyls (as Aroclor 1260) in human serum. There are no statistically significant differences in data obtained by these two quantifying techniques which were indicated by the paired t-test. For chlorinated pesticides, p = 0.053-0.62, and for polychlorinated biphenyls (as Aroclor 1260), p = 0.64. Analyte residues for the chlorinated pesticides ranged from 0.5 ppb for hexachlorobenzene (HCB) to 186 ppb for dichlorodiphenyldichloroethylene (DDE). Analyte residues for the polychlorinated biphenyls (as Aroclor 1260) ranged from 5-114 ppb. The absolute mean percent difference between the two quantifying techniques ranged from 0.06% for DDE to 8.06% for dieldrin (HEOD) among the chlorinated pesticides. The absolute mean percent difference between the two quantifying techniques for the polychlorinated biphenyls (as Aroclor 1260) was 3.4%. Peak area matching and linear regression were found to be comparable for quantifying these environmental residues in serum when the following conditions apply: 1) the concentration of the chlorinated pesticides is greater than or equal to 0.5 ppb (e.g., HCB, hexachlorocyclohexane (HCCH), oxychlordane (OC), heptachlor epoxide (HE), transnonachlor (TN), HEOD, and dichlorodiphenyltrichloroethane (DDT); 2) the concentration of the chlorinated pesticide is greater than or equal to 3 ppb (e.g., DDE); and 3) the total concentration of polychlorinated biphenyls (e.g., as Aroclor 1260) is greater than or equal to 5 ppb.

A Survey of Measured Levels and Dietary Sources of Selected Organochlorine Pesticide Residues and Metabolites in Human Sera from a Rural Population

We measured serum levels of 11 pesticide residues and metabolites in 85 rural-dwelling persons. In general, the serum levels increased with age, with males having slightly higher levels than females. Consumption of eggs from home-raised hens contributed substantially to increased serum concentrations of trans-nonachlor, heptachlor epoxide, and oxychlordane; consumption of home-grown root vegetables likewise contributed to increased serum concentrations of trans-nonachlor and oxychlordane. Health risks, if any, that may be attributable to these "background" levels of exposure remain to be fully characterized in this, and all other, affected populations.

Chlorinated Hydrocarbon Levels in Human Serum: Effects of Fasting and Feeding

Twenty healthy adult humans had serum samples drawn on four occasions within a 24-hr period: after a 12 hr overnight fast, 4-5 hr after a high fat breakfast, at midafternoon, and the next morning after another 12 hr fast. Nonfasting samples had 22% to 29% higher mean concentrations (p less than 0.05) than did fasting samples for polychlorinated biphenyls (PCBs, 4.81 vs 3.74 ng/g serum wt), hexachlorobenzene (HCB, 0.163 vs 0.134 ng/g serum wt), and p,p'-dichlorodiphenyl-dichloroethylene (p,p'-DDE, 6.74 vs 5.37 ng/g serum wt) measured by electron capture gas liquid chromatography. Total serum lipids were estimated from measurements of total cholesterol, free cholesterol, triglycerides, and phospholipids and were 20% higher in nonfasting samples than in fasting samples (7.05 g/L vs 5.86 g/L). When PCBs, HCB, and p,p'-DDE concentrations were corrected by total serum lipids, results from fasting and non-fasting samples were not statistically different. Because of the differences in these chlorinated hydrocarbon concentrations observed with different sample collection regimens, meaningful comparison of analytical results requires standardizing collection procedures or correcting by total serum lipid levels.

Half-life of Polychlorinated Biphenyls in Occupationally Exposed Workers

In 1977 and 1985, serum polychlorinated biphenyl (PCB) concentrations were determined for 58 workers in a Bloomington, Indiana, factory that used polychlorinated biphenyls (PCBs) in capacitor manufacture until 1977. Less chlorinated PCBs were quantitated as Aroclor 1242, and more highly chlorinated PCBs were quantitated as Aroclor 1254. The median half-life was 2.6 y for Aroclor 1242 and 4.8 y for Aroclor 1254. However, the half-life varied inversely with the initial serum concentration. This pattern may be a result of continued low-level exposure, variation in the time of exposure, or enzyme induction by PCBs.

Gas Chromatographic Determination of Polychlorinated Biphenyls (as Aroclor 1254) in Serum: Collaborative Study

A gas chromatographic-electron capture detection method for determining the concentration of polychlorinated biphenyls (PCBs) as Aroclor 1254 (AR 1254) in serum was evaluated through a 2-phase collaborative study. In Phase I, each collaborator's lot of Woelm silica gel (70-150 mesh) was evaluated for elution and recovery of AR 1254, which had been added in vitro at 25 ng/mL to a serum extract. In Phase II, each collaborator analyzed a series of bovine serum samples that contained the following: (1) in vitro-spiked AR 1254; (2) in vivo AR 1254 and 8 in vitro-spiked chlorinated hydrocarbons; (3) in vivo AR 1254 only; (4) 8 in vitro-spiked chlorinated hydrocarbons only; and (5) neither AR 1254 nor chlorinated hydrocarbons above the detection limit of the method. In Phase I, the average recovery of AR 1254 from silica gel for the 6 collaborators was 87.9 +/- 15.44% (mean +/- 1 SD; N = 18; range = 52.3-105.8%). In Phase II, the analysis of in vitro spikes of AR 1254 in serum at 8.58, 16.8, 41.8, and 84.3 ppb gave mean (means) interlaboratory recoveries of 89.0, 83.3, 79.4, and 76.9%, respectively, with within-laboratory (repeatability) relative standard deviations (RSDr) of 18.8, 20.5, 10.2, and 14.1%, respectively, and among-laboratory (reproducibility) relative standard deviations (RSDR) of 21.5, 21.1, 14.6, and 20.8%, respectively. The determination of in vivo AR 1254 in samples containing approximately 10, 25, 50, and 100 ng/mL of AR 1254 resulted in interlaboratory means of 10, 22, 39, and 79 ng/mL, respectively, with RSDr = 6.7, 9.7, 6.4, and 5.8%, respectively, and RSDR = 20.6, 16.0, 10.9, and 10.3%, respectively. The precision of the method for incurred AR 1254 showed a maximum RSDr of less than 10% and a maximum RSDR of less than 21% for a concentration range of 10-100 ng/mL. The accuracy of the method as demonstrated by the mean recovery of in vitro-spiked AR 1254 over a concentration range of 8.58-843 ng/mL was 82.2%. The method has been approved interim official first action.

Operation Everest II: Comparison of Four Instruments for Measuring Blood O2 Saturation

The bias and precision of four different methods for determining O2 saturation (SO2) were evaluated during a study of hypobaric hypoxia conducted with seven male subjects exposed progressively over a 40-day period to simulated altitudes from sea level (760 Torr) to 8,840 m (240 Torr). SO2 of arterial and mixed venous blood samples were measured with the Instrumentation Laboratory 282 CO-oximeter (CO-OX), the Radiometer ABL-300 (ABL), and the Lex-O2-Con-K (LEX). Noninvasive measurements of arterial SO2 were made with a Hewlett-Packard 47201A ear oximeter (EAR-OX). The CO-OX method was used as a secondary standard for comparison with the other methods because it has been validated against the classical Van Slyke method over a wide physiological range (Maas et al., Clin. Chim. Acta 29: 303-309, 1970). The LEX results most closely approximated but consistently underestimated those of the CO-OX: LEX = 0.93 CO-OX -0.86, standard error of the estimate (SEE) = 5.17, r = 0.98, n = 670. The ABL method appeared to combine two linear trends: for SO2 greater than 75%, ABL = 0.84 CO-OX +14.4, SEE = 1.77, r = 0.97, n = 369; less than 75%, ABL = 0.98 CO-OX +5.9, SEE = 4.44, r = 0.97, n = 486. The EAR-OX results were found to approximate those of the CO-OX at SO2 values only greater than 65%: EAR-OX = 1.07 CO-OX -6.12, SEE = 7.71, r = 0.98, n = 326.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum Pesticide Concentrations in Farming Cooperatives in Honduras

Determination of Selected Organochlorine Pesticides and Polychlorinated Biphenyls in Human Serum

A method is presented that can be used to determine the residue level of certain chlorinated pesticides and polychlorinated biphenyls (as Aroclor 1260) in serum. The method involves the following: (1) extraction of denatured serum with organic solvents; (2) elution of the organic extract through micro-Florisil columns to obtain two fractions; (3) acid treatment of the less polar Florisil fraction and its subsequent elution through deactivated silica gel to obtain two fractions; and (4) analysis of all three fractions using gas-liquid chromatography with electron capture detection. The method produced in vitro recoveries for 10 pesticides spiked in the range of 1-10.7 ppb of 50.4% to 121.6%, and in the range of 4.98-21 ppb, recoveries ranged from 47.7 to 112.6. In vivo "recoveries" of Aroclor 1260 averaged 104.8% and 92.3% for concentration levels of approximately 10 and approximately 30 ppb, respectively. The method could not be compared with the more commonly used hexane extraction technique because of the deleterious effect these extracts had on the gas chromatographic system.

Symptomatology During Hypoxic Exposure to Flame-retardant Chamber Atmospheres

Hypoxia was studied in 12 men during 63-h exposures to 17 and 13% O2, with the subjects serving as their own controls by repeating the measurements in 21% O2. All test atmospheres were contaminated with 0.9% CO2 to simulate the condition of living aboard submarines. The mean SaO2's were 97-98% in all conditions of 21% O2, 96% in 17% O2 (n.s.), and 92% in 13% O2 (P less than 0.05). The blood concentrations of 2,3-diphosphoglycerate were elevated in 13 and 17% O2 (P less than 0.05). Seventeen percent O2 did not cause significant symptoms of environmental stress; however, 13% O2 caused symptoms of acute mountain sickness in 5 of 12 men. In the last 7 h of exposure to 17% O2, reduction of the barometric pressure to 576 Torr reduced the ambient PO2 to 98 Torr (similar to the PO2 of 13% O2 at normobaric pressure). This induced symptoms of acute mountain sickness in 3 of 11 men. All symptomatology and physiologic changes were reversed during recovery in 21% O2. Monitoring devices indicated the presence of volatile organic contaminants at a mean concentration of 6.1 ppm in the chamber atmosphere. Combustion tests in the occupied chamber showed that flame propagation was retarded by lowering the O2 concentration from 21 to 13-17%. We conclude that men can live comfortably in a normobaric, flame-retardant atmosphere consisting of 17% O2-0.9% CO2-6.1 ppm volatile organic compounds-balance N2.

Use of Reference Pools to Compare the Qualitative and Quantitative Determination of Polychlorinated Biphenyls by Packed and Capillary Gas Chromatography with Electron Capture Detection. Part 1. Serum

Serum for reference pools of in vivo polychlorinated biphenyls (PCBs) was obtained from four goats that had received one dose (100 mg kg-1) of a selected technical Aroclor (AR) (1016, 1242, 1254 or 1260) and were allowed to recover for 30 d. These pools were used to assess the differences in an analytical method that determines and quantifies PCBs using packed-column gas chromatography (PCGC) (quantified on the basis of mean mass percent. data for grouped PCB peaks) and capillary-column gas chromatography (CCGC) (quantified on the basis of percent. composition data for specific congeners). With CCGC, results were statistically significantly different (p less than or equal to 0.0002) from results with PCGC for ARs 1016, 1242 and 1254 but not for AR 1260 (p = 0.23). When comparing these gas chromatographic methods using bovine serum spiked in vitro with the same ARs at 17-25 p.p.b., it was found that the methods were not statistically significantly different for any of the ARs (p = 0.30-0.92). Levels of serum PCB determined by the two methods for 12 persons, divided into two groups according to exposure, were compared using the paired t-test. Group 1 consisted of three persons with dietary and/or environmental exposure; one with dietary and/or environmental exposure in addition to occupational exposure dating back 20 years. Group 2 consisted of eight persons with recent occupational exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential Compounds for Monitoring Method Performance in the Determination of Selected Organochlorine Pesticides and Polychlorinated Biphenyls in Human Serum

Four compounds--2,2', 3,4'-tetrachlorobiphenyl, decachlorodiphenylether, 1,1-dichloro-2,2-bis(p-ethylphenyl)ethane, and dichlorobenzophenone--are recommended for monitoring the within-sample behavior of an analytical method that quantifies chlorinated pesticides and polychlorinated biphenyls (such as Aroclor 1260) in serum using packed column gas chromatography with electron capture detection. Percent recoveries of these surrogates averaged greater than 80%, except with dichlorobenzophenone, which had an average recovery of greater than 70%.

Operation Everest II: Neuromuscular Performance Under Conditions of Extreme Simulated Altitude

The force output of the ankle dorsiflexors was studied during a 40-day simulated ascent of Mt. Everest in a hypobaric chamber; both electrically activated and maximal voluntary contractions (MVCs) were employed. The purpose of this study was to establish whether, under conditions of progressive chronic hypoxia, there was a decrease in muscle force output and/or increased fatigability. We also attempted to identify the main site of any failure, i.e., central nervous system, neuromuscular junction, or muscle fiber. Muscle twitch torque (Pt), tetanic torque (Po), MVC torque, and evoked muscle compound action potential (M wave) were monitored during 205-s exercise periods in five subjects at three simulated altitudes (760, 335, and 282 Torr). All three types of torque measurement were well preserved at the three altitudes. In some subjects, the responses to stimuli interpolated during repeated MVCs provided evidence of "central" fatigue at altitude. In addition, the rate of fatigue during 20-Hz electrical stimulation was greater (P less than 0.01) at altitude and there was increased fatigability of the twitch (P less than 0.025); however, the M wave amplitude was maintained. We conclude that central motor drive becomes more precarious at altitude and is associated with increased muscle fatigue at low excitation frequencies; the latter is the result, in part, of chronic hypoxia and occurs in the muscle fiber interior because no impairment in neuromuscular transmission could be demonstrated.

Determination of Mirex in Human Blood Serum Containing Polychlorinated Biphenyls by Using Packed Column Gas Chromatography

An analytical method has been developed that uses electron capture/gas-liquid chromatography to determine Mirex in serum containing polychlorinated biphenyls (PCBs) (Aroclor 1260). With this method, 0.2 ppb Mirex can be determined in 4 mL serum that also contains 10 ppb PCBs. The method provides approximately 70% recovery of Mirex at 1.0 and 3.5 ppb. The coefficients of variation are 4.5 and 4.6% at 1.0 and 3.5 ppb, respectively. In a cooperative study with the Ohio Department of Health, the Centers for Disease Control used this method to determine the extent of exposure of Salem, OH, residents to Mirex. Confirmation of Mirex was obtained by using high resolution gas chromatography and high resolution mass spectrometry.

International Collaboration in a Cluster Investigation

Human Exposure to Polychlorinated Biphenyls in Greater New Bedford, Massachusetts: a Prevalence Study

A study was conducted in the community of Greater New Bedford, Massachusetts, from 1984 through 1987 to assess the prevalence of elevated levels of polychlorinated biphenyls (PCBs) in the serum of individuals aged 18 to 64 years who had resided in the area for at least 5 years. Eight hundred and forty subjects were interviewed, examined, and tested in a cross-sectional sample of the towns of Acushnet, Dartmouth, and Fairhaven and the city of New Bedford. Serum PCBs were measured to estimate the extent of human exposure. Because of documented environmental contamination by PCBs in the New Bedford area, and the practice of recreational fishing in the harbor for food, a significant number of persons with elevated serum PCB levels were expected to be identified. Instead, the prevalence of elevated serum PCBs in the sample was found to be typical of "unexposed" urban populations in the United States. Only 1.3% of the subjects had serum PCB levels greater than 30 ppb. The same percentage was observed among males (n = 391) and females (n = 449). The geometric means of PCB levels were 4.3 ppb among males (Range = 0.50-60.9) and 4.2 ppb among females (Range = 0.38-154). We conclude that the prevalence of elevated serum PCBs is low in the population of Greater New Bedford.

Problems Associated with Interferences in the Analysis of Serum for Polychlorinated Biphenyls

During a recent survey to determine serum concentrations of polychlorinated biphenyls (PCBs) among people living around New Bedford, MA, U.S.A., an unidentified contaminant precluded the quantification of some early eluting Webb and McCall peaks. Loss of data is estimated to have reduced reported serum levels by 12%. Efforts to identify the contaminant by gas chromatography with an electron-capture detector, a Hall electrolytic condutivity detector, and mass spectrometer were not successful. Researchers ascertained, however, that the contaminant is not a PCB, it does not contain halogens, but it may contain phthalates. Vacutainer tubes and closures for serum storage bottles are suspected sources of contamination.

Evidence of an Unusual Pattern of Polychlorinated Biphenyls in the Serum of Some Residents and Canines in Paoli, Pennsylvania

The present study uses gas liquid chromatography (GLC) electron capture detection with packed and capillary columns to detect polychlorinated biphenyls (PCBs) in serum samples from people living near the electric car repair and maintenance facility of the Southeastern Pennsylvania Transit Authority in Paoli, Pennsylvania. Most of the cohort surveyed had serum patterns similar to patterns for Aroclor 1260 (AR 1260); a small portion (3/89) had patterns indicative of an AR with higher chlorination (e.g., AR 1268). In addition to analyzing serum samples from humans, we also analyzed serum samples from canines (pets of some of the subjects). In general, the serum pattern for canines was less descriptive for AR 1260 than the pattern for humans; however, the pattern for several canines (9/16) was that of the higher chlorinated PCBs (e.g., AR 1268). By using mass spectrometry and capillary column GLC, we confirmed the presence of high molecular weight polychlorinated congeners in both human and animal samples. We were not able to show a statistically significant relationship between serum patterns of PCBs in canines and their owners or between canines and certain behavioral traits (e.g., runs free, retrieves, hours outside, hours inside). However, the correlation between PCBs quantified as AR 1268 and canines' residence time was statistically significant.

Environmental Equity and Pesticide Exposure

Although people of color and low-income groups bear a disproportionate share of the health risks from exposure to pesticides, research attention has been meager, and data on acute and chronic health effects related to their toxic exposures are generally lacking. Increased resources are needed both to study this issue and to mitigate problems already identified. People of color should be a major research focus, with priority on long-term effects, particularly cancer, neurodevelopmental and neurobehavioral effects, long-term neurological dysfunction, and reproductive outcome. Suitable populations at high risk that have not been studied include noncertified pesticide applicators and seasonal and migrant farm workers, including children.

Determination of Polychlorinated Biphenyl Levels in the Serum of Residents and in the Homogenates of Seafood from the New Bedford, Massachusetts, Area: a Comparison of Exposure Sources Through Pattern Recognition Techniques

We measured the residues of polychlorinated biphenyls (PCBs) in the serum of 23 residents of the New Bedford, Massachusetts, area and from two homogenates each of bluefish and lobsters from the same area. We used congener-specific and total Aroclor quantitative approaches, both of which involved gas chromatography with electron capture detection. Using gas chromatography mass spectrometry (electron ionization mode), we confirmed the presence of PCBs in the combined serum samples and in the aliquots of bluefish and lobsters. In measuring the PCB levels in serum, we found good agreement between the two electron capture detector approaches (r > or = 0.97) when the serum of specific congeners was compared to total Aroclor. We used univariate and multivariate quality control approaches to monitor these analyses. Analytical results for bluefish showed a better agreement between the two techniques than did those for lobsters; however, the small number of samples precluded any statistical comparison. We also measured levels of chlorinated pesticides in the serum samples of two groups of New Bedford residents, those with low PCB levels (< 15 ng/ml) and those with high PCB levels (> or = 15 ng/ml). We found that residents with high PCB levels also tended to have higher levels of hexachlorobenzene (HCB) and 1,1-dichloro-2,2-di-(p-chlorophenyl) ethylene (p,p'-DDE). The higher concentration of all three analytes appears to be influenced by employment in the capacitor industry, by seafood consumption, or both. Using Jaccard measures of similarity and principal component analysis we compared the gas chromatographic patterns of PCBs found in the serum of New Bedford area residents with high serum PCBs with the patterns found in homogenates of lobsters (inclusive of all edible portions except the roe), in homogenates of bluefish fillets taken from local waters, and in serum from goats fed selected technical Aroclors (e.g. Aroclors 1016, 1242, 1254, or 1260). The patterns found in human serum samples were similar to the patterns found in lobster homogenates. Both of these patterns closely resembled patterns found in the serum samples of the goat fed aroclor 1254, as demonstrated by both pattern recognition techniques. In addition, the chromatographic patterns of human serum and of lobsters and bluefish homogenates all indicated the presence of PCBs more characteristic of Aroclors 1016 or 1242.

Relation of a Seafood Diet to Mercury, Selenium, Arsenic, and Polychlorinated Biphenyl and Other Organochlorine Concentrations in Human Milk

Human transition milk was sampled from 88 mothers at the Faroe Islands, where the seafood diet includes pilot whale meat and blubber. Milk mercury concentrations (median, 2.45 micrograms/liter) were significantly associated with mercury concentrations in cord blood and with the frequency of pilot whale dinners during pregnancy. Milk selenium concentrations (mean, 19.1 micrograms/liter) correlated significantly with concentrations in cord blood but not with seafood consumption. Arsenic concentrations were very low. Twenty-four of the milk samples were separated into four pools based on fish intake and milk mercury concentrations. The polychlorinated biphenyl (PCB) concentrations (1.8-3.5 micrograms/g lipid) were high and mainly due to congener numbers 153, 180, and 138. One pool contained a congener 77 concentration of 1380 ppt, which is the highest ever reported in a human specimen for a coplanar PCB. The highest PCB concentrations were seen in the pools from women who had eaten frequent whale dinners and whose milk contained high mercury concentrations. The concentrations of chlorinated dibenzo-p-dioxins and furans were not similarly elevated. Given the advantages associated with breast-feeding, advice to nursing mothers in this population should take into regard the possible risks associated with long-term exposure to milk contaminants.

Selected Analytical Methods Used at the Centers for Disease Control and Prevention for Measuring Environmental Pollutants in Serum

Blood serum is one of the more viable matrices used in assessing exposure to persistent environmental contaminants or their metabolites, especially those that are lipophilic. Analytic methods currently in use for this matrix usually involve liquid/liquid extraction followed by adsorption chromatography as a cleanup step, and low- or high-resolution gas chromatography with either electron-capture or mass spectrometric detection. The traditional analytic methods are labor intensive, have low sample throughput, and use excessive amounts of solvents and reagents. Two analytic approaches that address the requirements of modern laboratories more effectively are: 1) solid-phase extraction (SPE), used to analyze serum for several classes of compounds of environmental concern (e.g., polychlorinated biphenyls [PCBs], persistent pesticides, dioxins, furans, and coplanar polychlorinated biphenyls [CPCBs]), and 2) fast chromatography with a two-dimensional gas chromatographic system, which can be used in the determinative step for these types of analytes.

Reference Range Data for Assessing Exposure to Selected Environmental Toxicants

We analyzed blood and urine specimens from 32 charter boat captains, anglers, and spouses from both groups, who reportedly ate fish from Lakes Michigan, Huron, or Erie, for selected environmental toxicants. The toxicants measured in serum were polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), coplanar polychlorinated biphenyls, other polychlorinated biphenyls (PCBs), and persistent pesticides. Nonpersistent pesticides and elements were measured in urine; and elements were measured in blood. Internal dose levels of these toxicants will be compared to reference range data that we have compiled. These reference range data will be used to ascertain the exposure status of individuals or groups within this study.

An Improved Analysis for Chlorinated Pesticides and Polychlorinated Biphenyls (PCBs) in Human and Bovine Sera Using Solid-phase Extraction

Chlorinated pesticides and polychlorinated biphenyls (PCBs) remain public health concerns because of their unresolved health impact and their persistence in humans. Current epidemiological studies of cancer, non-Hodgkins lymphoma, and endocrine disruption in National Center for Environmental Health (NCEH) laboratories require exposure assessment of many analytes in thousands of people. Previous methods of analyzing pesticides and PCBs in serum have proven inadequate for timely processing of the number of samples required for epidemiological studies. A new method that involves solid-phase extraction (SPE) and cleanup followed by dual-column gas chromatographic separation and electron capture detection has been developed. Nine surrogate compounds were added to the serum prior to sample workup to provide quality assurance for the SPE steps. These surrogates mimic the chemistry of the analytes in the extraction, cleanup, and gas chromatographic analysis steps. To increase selectivity, extracts were injected onto two gas chromatographs with different capillary columns, a DB-1701 and a DB-5. Recoveries of 17 pesticides, 28 PCB congeners, and one polybrominated biphenyl congener ranged from 40 to 80%. Recoveries from this procedure were found to be similar to those from the previously used liquid-liquid extraction method. Correlation of analyte and surrogate recoveries were compared to examine the ruggedness of the technique. The SPE method was found to provide improved sample throughput by a factor of 15.

Blood Oxygen Saturation Measured in the Presence of a Mixture of Cyclopropane, Sulfur Hexafluoride, and Ethane

Some anesthetic gases interfere with the determination of blood O2 values. We evaluated, for its potential for such interference, a gas mixture containing (v/v) approximately 70% ethane, 20% sulfur hexafluoride, and 10% cyclopropane, as is currently used in trace amounts to determine ventilation-perfusion (v/Q) ratios. Normal human blood samples were first tonometered with control gas mixtures containing (v/v): (1) 20.9% O2, 0.04% CO2; (2) 16.0% O2, 4.0% CO2; and (3) 9.9% O2, 7.8% CO2. A large quantity (20.9%) of the v/Q mixture was blended experimentally into the control mixture and the tonometry repeated. The entire experiment was then repeated substituting pure N2 for the v/Q mixture as a dilution control. O2 values were determined by three methods: (1) a polarographic electrode, ABL-300 (ABL); (2) a spectrophotometric method, Co-Oximeter (COOX); and (3) a galvanic cell, Lex-O2-Con (LEX). The v/Q gas mixture lowered significantly all measured LEX values by 2.5-3.6 saturation percent (sat%), but showed no effect, dilution or otherwise, on the O2 values determined by the COOX and ABL methods. The N2 dilution lowered the LEX values by an average of only 0.9 sat%; the ABL and the COOX were approximately 0.6 sat% lower. We therefore suggest that, if any of these O2 measurement methods are used in the presence of the v/Q mixture, baseline O2 values should be determined both before and after injection of the mixture into the bloodstream, prior to performing other experimental manipulations. The difference between the two values, if any, can then be used to interpret subsequent results.

Preliminary Investigation of the Use of Dried-blood Spots for the Assessment of in Utero Exposure to Environmental Pollutants

We determined the concentration of dichlorodiphenyldichloroethylene (p,p'-DDE) in dried-blood spot specimens from 2-day-old infants from rural Texas who had never been breast fed. Anonymous, residual whole blood spots on filter paper, previously used for routine newborn screening procedures, were soaked in a phosphate buffer, extracted with an organic solvent, and eluted through silica gel. The concentrated eluates were analyzed by capillary gas chromatography with electron capture detection (ECD). The blood collected from 10 newborns was analyzed and found to contain DDE concentrations ranging from 0.13 to 1.87 pg/microliter with a mean of 0.72 pg/microliter. One of the 10 newborns had a whole blood DDE concentration of 1.87 pg/microliter, which was greater than the concentration of 1.34 pg/microliter in a freshly drawn sample from an adult donor whose blood serum was shown to contain DDE. With improvement in detection limits, this approach has the potential to displace the analyses of mothers' blood (as a surrogate indicator of infants' exposures) and cord blood as standard procedures for determining the newborns' body burden of environmental pollutants.

Profiles of Great Lakes Critical Pollutants: a Sentinel Analysis of Human Blood and Urine. The Great Lakes Consortium

To determine the contaminants that should be studied further in the subsequent population-based study, a profile of Great Lakes (GL) sport fish contaminant residues were studied in human blood and urine specimens from 32 sport fish consumers from three Great Lakes: Lake Michigan (n = 10), Lake Huron (n = 11), and Lake Erie (n = 11). Serum was analyzed for 8 polychlorinated dioxin congeners, 10 polychlorinated furan congeners, 4 coplanar and 32 other polychlorinated biphenyl (PCB) congeners, and 11 persistent chlorinated pesticides. Whole blood was analyzed for mercury and lead. Urine samples were analyzed for 10 nonpersistent pesticides (or their metabolites) and 5 metals. One individual was excluded from statistical analysis because of an unusual exposure to selected analytes. Overall, the sample (n = 31) consumed, on average, 49 GL sport fish meals per year for a mean of 33 years. On average, the general population in the GL basin consume 6 meals of GL sport fish per year. The mean tissue levels of most persistent, bioaccumulative compounds also found in GL sport fish ranged from less than a twofold increase to that of PCB 126, which was eight times the selected background levels found in the general population. The overall mean total toxic equivalent for dioxins, furans, and coplanar PCBs were greater than selected background levels in the general population (dioxins, 1.8 times; furans, 2.4 times; and coplanar PCBs, 9.6 times). The nonpersistent pesticides and most metals were not identified in unusual concentrations. A contaminant pattern among lake subgroups was evident. Lake Erie sport fish consumers had consistently lower contaminant concentrations than consumers of sport fish from Lake Michigan and Huron. These interlake differences are consistent with contaminant patterns seen in sport fish tissue from the respective lakes; GL sport fish consumption was the most likely explanation for observed contaminant levels among this sample. Frequent consumers of sport fish proved to be effective sentinels for identifying sport fish contaminants of concern. In the larger study to follow, serum samples will be tested for PCBs (congener specific and coplanar), DDE, dioxin, and furans.

Analysis of a Mixture of Polychlorinated Biphenyls and Chlorinated Pesticides in Human Serum by Column Fractionation and Dual-column Capillary Gas Chromatography with Electron Capture Detection

An analytical method is presented for precise identification and quantitation of 29 specific polychlorinated biphenyl (PCB) congeners and 15 chlorinated pesticides in human serum. Analyte surrogates PCB 30, PCB 204, 2,2',4,4',5,5'-hexabromo-biphenyl, perthane, alpha-hexachlorocyclohexane, and dichlorobenzophenone were added to each sample. The serum was extracted with an organic solvent and separated by adsorption chromatography into 3 elution fractions for high-resolution gas chromatographic analysis. Each fraction was analyzed by dual-column capillary chromatography followed by electron capture detection. Two capillary columns, DB-5 and DB-1701, with different polarities were used to increase selectivity for each analyte. Quantitation was performed by selecting 2 sets of calibration standard mixtures and 1,2-dichloronaphthalene as an internal standard. Mean recoveries ranged from 39 to 126% for selected analytes and from 31 to 88% for surrogates. Detection limits for specific congeners and pesticides are reported. Typical chromatographic profiles of calibration standard mixtures, as well as a human sample, are illustrated. Verification of each analyte is assessed, and results of analyses of selected human samples and quality control criteria used to ensure data validity also are presented.

Serum Concentrations of Organochlorine Compounds and the Subsequent Development of Breast Cancer

A nested case-control study was conducted to examine the association between serum concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), the primary metabolite of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and polychlorinated biphenyls (PCBs) and the development of breast cancer up to 20 years later. Cases (n = 346) and controls (n = 346) were selected from cohorts of women who donated blood in 1974, 1989, or both, and were matched on age, race, menopausal status, and month and year of blood donation. Analyses were stratified by cohort participation because median DDE and PCB concentrations among the controls were 59 and 147% higher in 1974 than 1989, respectively. Median concentrations of DDE were lower among cases than controls in both time periods [11.7% lower in 1974 (P = 0.06) and 8.6% lower in 1989 (P = 0.41)]. Median concentrations of PCBs were similar among cases and controls [P = 0.21 for 1974 and P = 0.37 for 1989 (Wilcoxon signed rank test)]. The risk of developing breast cancer among women with the highest concentrations of DDE was roughly half that among women with the lowest concentrations, whether based on concentrations in 1974 [odds ratio (OR), 0.50; 95% confidence interval (CI), 0.27-0.89; P(trend) = 0.02] or in 1989 (OR, 0.53; 95% CI, 0.24-1.17; P(trend) = 0.08). The associations between circulating concentrations of PCBs and breast cancer were less pronounced but still in the same direction (1974: OR, 0.68; 95% CI, 0.36-12.9; P(trend) = 0.2; and 1989: OR, 0.73; 95% CI, 0.37-1.46; P(trend) = 0.6). Adjustment for family history of breast cancer, body mass index, age at menarche or first birth, and months of lactation did not materially alter these associations. These associations remained consistent regardless of lactation history and length of the follow-up interval, with the strongest inverse association observed among women diagnosed 16-20 years after blood drawing. Results from this prospective, community-based nested case-control study are reassuring. Even after 20 years of follow-up, exposure to relatively high concentrations of DDE or PCBs showed no evidence of contributing to an increased risk of breast cancer.

Pancreatic Cancer and Serum Organochlorine Levels

Occupational exposure to p,p'-dichlorodiphenyltrichloroethane (DDT) has been associated with increased pancreatic cancer risk. We measured organochlorine levels in serum obtained at the study enrollment from 108 pancreatic cancer cases and 82 control subjects aged 32-85 years in the San Francisco Bay Area between 1996 and 1998. Cases were identified using rapid case-ascertainment methods; controls were frequency-matched to cases on age and sex via random digit dial and random sampling of Health Care Financing Administration lists. Serum organochlorine levels were adjusted for lipid content to account for variation in the lipid concentration in serum between subjects. Median concentrations of p,p'-dichlorodiphenyldichloroethylene (DDE, 1290 versus 1030 ng/g lipid; P = 0.05), polychlorinated biphenyls (PCBs; 330 versus 220 ng/g lipid; P<0.001), and transnonachlor (54 versus 28 ng/g lipid; P = 0.03) were significantly greater among cases than controls. A significant dose-response relationship was observed for total PCBs (P for trend <0.001). Subjects in the highest tertile of PCBs (> or =360 ng/g lipid) had an odds ratio (OR) of 4.2 [95% confidence interval (CI) = 1.8-9.4] compared to the lowest tertile. The OR of 2.1 for the highest level of p,p'-DDE (95% CI = 0.9-4.7) diminished (OR = 1.1; 95% CI = 0.4-2.8) when PCBs were included in the model. Because pancreatic cancer is characterized by cachexia, the impact of this on the serum organochlorine levels in cases is difficult to predict. One plausible effect of cachexia is bioconcentration of organochlorines in the diminished lipid pool, which would lead to a bias away from the null. To explore this, a sensitivity analysis was performed assuming a 10-40% bioconcentration of organochlorines in case samples. The OR associated with PCBs remained elevated under conditions of up to 25% bioconcentration.

Measurement of PCBs, DDE, and Hexachlorobenzene in Cord Blood from Infants Born in Towns Adjacent to a PCB-contaminated Waste Site

There are limited data on the concentrations of common contaminants--polychlorinated biphenyls (PCBs), dichlorodiphenyl dichloroethene (pp'-DDE) and hexachlorobenzene (HCB)--in umbilical cord blood. Cord blood provides the primary direct measure of prenatal exposure to these contaminants, the key determinant of PCBs' neurodevelopmental toxicities. The objective of this study was to characterize cord blood levels of PCBs, pp'-DDE, and HCB among 751 infants who were born between 1993 and 1998 to mothers residing adjacent to a PCB-contaminated harbor in New Bedford, Massachusetts, and for whom the neurodevelopmental toxicities of these compounds are being studied. We refined standard analytic methods to optimize the sensitivity and precision of trace-level PCB, p,p'-DDE, and HCB measurements in blood. Using these methods, we measured the concentrations of 51 individual PCBs, their sum (sum(PCB)), p,p'-DDE, and HCB in cord serum. With correction for background contamination, the respective mean+/-SD cord serum concentrations of sum(PCB), p,p'-DDE, and HCB were 0.54+/-0.83, 0.48+/-0.94, and 0.03+/-0.04 ng/g serum. These concentrations were generally lower than those in most of the few published studies with congener-specific measures of PCBs in cord blood. However, for less-chlorinated PCB congeners (e.g., congeners 99 and 118), study samples had concentrations comparable to those in other populations, including groups at risk for high dietary PCB exposure. Of note, the contaminated harbor sediment has a relatively high proportion of less-chlorinated PCB congeners. Thus, although the sum(PCB) in study infants was not higher than concentrations in infants studied elsewhere, the relative predominance of less-chlorinated congeners was generally consistent with the characteristics of the contaminated site.

Utilization of Umbilical Cords to Assess in Utero Exposure to Persistent Pesticides and Polychlorinated Biphenyls

In support of a study to relate developmental and cognitive effects with prenatal exposure to selected environmental toxicants, we developed and applied an analytical method to determine the concentration of two persistent pesticides, hexachlorobenzene (HCB) and p,p'-dichlorodiphenyldichloroethylene (DDE), and 32 specific polychlorinated biphenyl (PCB) congeners in 316 umbilical cords taken in 1986-1987 from women of the Faroe Islands. The analytical method consisted of homogenization of the cords, partitioning, microsilica gel column chromatography for clean-up, and dual-column capillary gas chromatography (DB-5 and DB-1701) with electron capture detection. Several quality control parameters were followed to monitor the performance of the method. Important criteria used before reporting unknown data were the recovery of in vitro-spiked analytes from a bovine umbilical cord (BUC) and the percentage lipid obtained for a Certified Reference Material (CRM)-350 of mackerel oil (MO). Recoveries of analytes that had been spiked at two concentration ranges (0.26-0.95 ng/g whole weight; 0.35-2.42 ng/g whole weight) into bovine cords ranged from 38.5% to 158% and from 50.4% to 145%, respectively, with a median recovery of 77.7%. Measurement of the percentage lipid for CRM-350 ranged from 73.8% to 107% with a median lipid value of 96.0%. The most prevalent analytes detected (%) in unknown umbilical cords were HCB (100), DDE (100), Ballschmiter/Zell PCBs 153 (100), 138 (98), 180 (98), 170 (93), 118 (88), 187 (86), and 146 (83), with corresponding median concentrations (ng/g whole weight) of 0.17, 1.19, 0.38, 0.30, 0.17, 0.11, 0.12, 0.09, and 0.07, respectively. Total PCB--sum of all measurable PCB congeners--had a median concentration of 1.37 ng/g whole weight. The analytes, which were very low in lipid content were also quantified on a lipid-adjusted basis, which provided an analytical challenge in these umbilical cord samples. The gravimetrically measured lipids in the human specimens ranged from 0.01% to 1.43% (median of 0.18%). In the pooled BUCs, our lipid measurements varied from 0.05% to 0.33% with a median value of 0.13%. The utility of using the umbilical cord as a matrix to assess in utero exposure to persistent environmental pollutants, compared with the use of umbilical cord blood or mother's blood, is worthy of debate.

Neurobehavioral Deficits Associated with PCB in 7-year-old Children Prenatally Exposed to Seafood Neurotoxicants

Prenatal exposure to polychlorinated biphenyls (PCBs) was examined by analysis of cord tissue from 435 children from a Faroese birth cohort. Analysis of 50 paired cord blood samples showed excellent correlation with the cord tissue concentration (r=.90). Among 17 neuropsychological outcomes determined at age 7 years, the cord PCB concentration was associated with deficits on the Boston Naming Test (without cues, two-tailed P=.09 not adjusted for mercury; with cues, P=.03), the Continuous Performance Test reaction time (P=.03), and, possibly, on long-term recall on the California Verbal Learning Test (P=.15). The association between cord PCB and cord-blood mercury (r=.42) suggested possible confounding. While no PCB effects were apparent in children with low mercury exposure, PCB-associated deficits within the highest tertile of mercury exposure indicated a possible interaction between the two neurotoxicants. PCB-associated increased thresholds were seen at two of eight frequencies on audiometry, but only on the left side, and no deficits occurred on evoked potentials or contrast sensitivity. The limited PCB-related neurotoxicity in this cohort appears to be affected by concomitant methylmercury exposure.

Thermoregulated Expression of Virulence Factors in Plant-associated Bacteria

Pathogenic bacteria with habitats inside and outside a given host react to changes in environmental parameters by synthesizing gene products specifically needed during pathogenic or saprophytic growth. Temperature effects have been investigated in detail for pathogens of warm-blooded hosts, and major principles governing the temperature-sensing mechanism have been uncovered. Generally, transcription of virulence genes in these pathogens is induced at higher temperatures (37-41 degrees C), which are typical for body cavities and host tissues. However, effects of temperature on virulence determinants in plant pathogenic bacteria have not been focused on in detail. Interestingly, almost all virulence genes of plant pathogenic bacteria studied with respect to temperature exhibit increased transcription at temperatures well below the respective growth optima. This includes virulence determinants such as those directing bacteria-to-plant gene transfer, plant cell-wall-degrading enzymes, phytotoxins, ice nucleation activity, exopolysaccharide production, and the type III protein secretion machinery. Although many of the studied phytopathogens cause "cold-weather" diseases, the ecological rationale for this phenomenon remains to be studied in detail. This mini-review summarizes our current knowledge on thermoregulation of cellular processes taking place in bacterial phytopathogens in response to temperature changes. Since the temperature range of interest is different from that relevant to pathogens of mammals, one envisions novel principles of thermo-sensing in bacteria interacting with plants.

The Phytoalexin-inducible Multidrug Efflux Pump AcrAB Contributes to Virulence in the Fire Blight Pathogen, Erwinia Amylovora

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.

NorM, an Erwinia Amylovora Multidrug Efflux Pump Involved in in Vitro Competition with Other Epiphytic Bacteria

Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.

Bluegill (Lepomis Macrochirus) Vitellogenin: Purification and Enzyme-linked Immunosorbent Assay for Detection of Endocrine Disruption by Papermill Effluent

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.

Selective Transport Systems Mediate Sequestration of Plant Glucosides in Leaf Beetles: a Molecular Basis for Adaptation and Evolution

Chrysomeline larvae respond to disturbance and attack by everting dorsal glandular reservoirs, which release defensive secretions. The ancestral defense is based on the de novo synthesis of monoterpene iridoids. The catabolization of the host-plant O-glucoside salicin into salicylaldehyde is a character state that evolved later in two distinct lineages, which specialized on Salicaceae. By using two species producing monoterpenes (Hydrothassa marginella and Phratora laticollis) and two sequestering species (Chrysomela populi and Phratora vitellinae), we studied the molecular basis of sequestration by feeding the larvae structurally different thioglucosides resembling natural O-glucosides. Their accumulation in the defensive systems demonstrated that the larvae possess transport systems, which are evolutionarily adapted to the glycosides of their host plants. Minor structural modifications in the aglycon result in drastically reduced transport rates of the test compounds. Moreover, the ancestral iridoid-producing leaf beetles already possess a fully functional import system for an early precursor of the iridoid defenses. Our data confirm an evolutionary scenario in which, after a host-plant change, the transport system of the leaf beetles may play a pivotal role in the adaptation on new hosts by selecting plant-derived glucosides that can be channeled to the defensive system.

Iridoid Biosynthesis in Chrysomelina Larvae: Fat Body Produces Early Terpenoid Precursors

Larvae of the Chrysomelina species Phaedon cochleariae and Gastrophysa viridula produce monoterpenoids (iridoids) to defend themselves against predatory attacks by presenting the toxins upon attack as droplets on the top of nine pairs of dorsal glands. Although the conversion of 8-hydroxygeraniol-8-O-beta-d-glucoside into the iridoids in the glandular reservoir has been studied in detail, the synthesis of the glucosidically bound precursor received only limited attention. We compared larvae of the two iridoid producing species with those of Chrysomela populi, a sequestering species producing salicylaldehyde, in terms of the key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and isoprenyl diphosphate synthases involved in the biosynthesis of the iridoid precursor. Increased HMGR transcript abundance, high HMGR activity and accumulation of geraniol indicating geranyl diphosphate synthase activity was observed only in the fat body of the iridoid producing larvae in comparison to other larval tissues and to the tested tissues of C. populi. These results correlate with the identification of glucosidically bound 8-hydroxygeraniol in the fat body of the iridoid producers. We suggest that in P. cochleariae and G. viridula glucosidically bound 8-hydroxygeraniol is produced by the fat body and transferred via the hemolymph into the glandular reservoir for further conversion into iridoids.

Thioglycosides As Inhibitors of HSGLT1 and HSGLT2: Potential Therapeutic Agents for the Control of Hyperglycemia in Diabetes

The treatment of diabetes has been mainly focused on maintaining normal blood glucose concentrations. Insulin and hypoglycemic agents have been used as standard therapeutic strategies. However, these are characterized by limited efficacy and adverse side effects, making the development of new therapeutic alternatives mandatory. Inhibition of glucose reabsorption in the kidney, mediated by SGLT1 or SGLT2, represents a promising therapeutic approach. Therefore, the aim of the present study was to evaluate the effect of thioglycosides on human SGLT1 and SGLT2. For this purpose, stably transfected Chinese hamster ovary (CHO) cells expressing human SGLT1 and SGLT2 were used. The inhibitory effect of thioglycosides was assessed in transport studies and membrane potential measurements, using alpha-methyl-glucoside uptake and fluorescence resonance energy transfer, respectively. We found that some thioglycosides inhibited hSGLT more strongly than phlorizin. Specifically, thioglycoside I (phenyl-1'-thio-beta-D-glucopyranoside) inhibited hSGLT2 stronger than hSGLT1 and to a larger extent than phlorizin. Thioglycoside VII (2-hydroxymethyl-phenyl-1'-thio-beta-D-galacto-pyranoside) had a pronounced inhibitory effect on hSGLT1 but not on hSGLT2. Kinetic studies confirmed the inhibitory effect of these thioglycosides on hSGLT1 or hSGLT2, demonstrating competitive inhibition as the mechanism of action. Therefore, these thioglycosides represent promising therapeutic agents for the control of hyperglycemia in patients with diabetes.

Congener Profiles of Occupational PCB Exposure Versus PCB Exposure from Fish Consumption

The composition of polychlorinated biphenyl (PCB) congeners in serum samples is compared between a cohort previously exposed to PCBs from working at a capacitor plant (n=180) and a cohort of Great Lakes sport-caught fish eaters (n=217). Fourteen congeners were measured in both samples. A multiple logistic regression model differentiating the two groups as a function of relative proportions amongst congeners 74, 138, 153, 180, and 201 correctly classifies more than 99% of the people (395/397); higher proportions of congeners 74, 153, and 201 characterize capacitor plant workers, while higher proportions of congeners 138 and 180 characterize fish eaters. The pattern is driven by the relative amounts of 74+153+201 compared to 138+180; all of the fish eaters, but only 5% of the capacitor plant workers, have a greater percent of 138+180 than 74+153+201. Consideration of combinations of congener levels and their relative proportions is relevant to tracking route of exposure and may also be relevant to modeling effects on health outcomes.

Implication of HMGR in Homeostasis of Sequestered and De Novo Produced Precursors of the Iridoid Biosynthesis in Leaf Beetle Larvae

Insects employ iridoids to deter predatory attacks. Larvae of some Chrysomelina species are capable to produce those cyclopentanoid monoterpenes de novo. The iridoid biosynthesis proceeds via the mevalonate pathway to geranyl diphospate (GDP) subsequently converted into 8-hydroxygeraniol-8-O-beta-D-glucoside followed by the transformation into the defensive compounds. We tested whether the glucoside, its aglycon or geraniol has an impact on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key regulatory enzyme of the mevalonate pathway and also the iridoid biosynthesis. To address the inhibition site of the enzyme, initially a complete cDNA encoding full length HMGR was cloned from Phaedon cochleariae. Its catalytic portion was then heterologously expressed in Escherichia coli. Purification and characterization of the recombinant protein revealed attenuated activity in enzyme assays by 8-hydroxygeraniol whereas no effect has been observed by addition of the glucoside or geraniol. Thus, the catalytic domain is the target for the inhibitor. Homology modeling of the catalytic domain and docking experiments demonstrated binding of 8-hydroxygeraniol to the active site and indicated a competitive inhibition mechanism. Iridoid producing larvae are potentially able to sequester glucosidically bound 8-hydroxygeraniol whose cleavage of the sugar moiety results in 8-hydroxygeraniol. Therefore, HMGR may represent a regulator in maintenance of homeostasis between de novo produced and sequestered intermediates of iridoid metabolism. Furthermore, we demonstrated that HMGR activity is not only diminished in iridoid producers but most likely prevalent within the Chrysomelina subtribe and also within the insecta.

A Versatile Transport Network for Sequestering and Excreting Plant Glycosides in Leaf Beetles Provides an Evolutionary Flexible Defense Strategy

The larval defenses of chrysomeline leaf beetles comprise components that are either synthesized de novo or sequestered from their food plants. Both biosynthetic modes are based on glucosides that serve as substrates and forms of transport. The defensive glands import the compounds through highly selective glucoside transporters from a circulating pool in the hemolymph. Here we address the selectivity of the different transport systems with larvae of Chrysomela populi, an obligate sequestering species, and with larvae of Phaedon cochleariae, producing monoterpene [corrected] iridoids. Both species possess an interconnected network of transport systems for uptake and excretion. The glucosides are imported by the gut membrane with low selectivity. Their excretion by the Malpighian tubules is similarly unselective, but the uptake of the glucosides from the hemolymph into the defensive system is specific. Only the genuine glucoside precursors made de novo or sequestered from the plant are imported. The successful combination of the precursor-adapted pathways of excretion and defense has probably allowed many leaf beetle species to adaptively radiate onto, and coevolve with plants that offer appropriate glucoside precursors.

Always Being Well Prepared for Defense: the Production of Deterrents by Juvenile Chrysomelina Beetles (Chrysomelidae)

In response to herbivores, plants produce a variety of natural compounds. Many beetle species have developed ingenious strategies to cope with these substances, including colonizing habitats not attractive for other organisms. Leaf beetle larvae of the subtribe Chrysomelina, for example, sequester plant-derived compounds and use them for their own defense against predators. Using systematically modified structural mimics of plant-derived glucosides, we demonstrated that all tested Chrysomelina larvae channel compounds from the gut lumen into the defensive glands, where they serve as intermediates in the synthesis of deterrents. Detailed studies of the sequestration process revealed a functional network of transport processes guiding phytochemicals through the larval body. The initial uptake by the larvae's intestine seems to be fairly unspecific, which contrasts sharply with the specific import of precursors into the defensive glands. The Malpighian tubules and hind-gut organs facilitate the rapid clearing of body fluid from excess or unusable compounds. The network exists in both sequestering species and species producing deterrents de novo. Transport proteins are also required for de novo synthesis to channel intermediates from the fat body to the defensive glands for further conversion. Thus, all the tools needed to exploit host plants' chemistry by more derived Chrysomelina species are already developed by iridoid-de novo producers. Early intermediates from the iridoid-de novo synthesis which also can be sequestered are able to regulate the enzyme activity in the iridoid metabolism.

Associations of Polychlorinated Biphenyl Exposure and Endogenous Hormones with Diabetes in Post-menopausal Women Previously Employed at a Capacitor Manufacturing Plant

There is an increasing body of literature showing associations of organochlorine exposure with risk of diabetes and insulin resistance. Some studies suggest that associations differ by gender and that diabetes risk, in turn, may be affected by endogenous steroid hormones. This report examines the relationships of serum PCBs and endogenous hormones with history of diabetes in a cohort of persons previously employed at a capacitor manufacturing plant. A total of 118 women were post-menopausal with complete data, of whom 93 were not using steroid hormones in 1996, at the time of examination, which included a survey of exposure and medical history, height, weight and collection of blood and urine for measurements of lipids, liver function, hematologic markers and endogenous hormones. This analysis examines relationships of serum polychlorinated biphenyls (PCBs), work exposure and endogenous hormones with self-reported history of diabetes after control for potential confounders. All PCB exposure groups were significantly related to history of diabetes, but not to insulin resistance as measured by the homeostatic model assessment of insulin resistance (HOMA-IR) in non-diabetics. Diabetes was also independently and inversely associated with follicle stimulating hormone (FSH), dehydroepiandrosterone sulfate (DHEAS) and triiodothyronine (T3) uptake. HOMA-IR was positively associated with body mass index (BMI) and C-reactive protein (CRP) and inversely associated with sex hormone binding globulin (SHBG) and T3 uptake after control for PCB exposure. Possible biologic mechanisms are discussed. This study confirms previous reports relating PCB exposure to diabetes and suggests possible hormonal pathways deserving further exploration.

Precise RNAi-mediated Silencing of Metabolically Active Proteins in the Defence Secretions of Juvenile Leaf Beetles

Allomones are widely used by insects to impede predation. Frequently these chemical stimuli are released from specialized glands. The larvae of Chrysomelina leaf beetles produce allomones in gland reservoirs into which the required precursors and also the enzymes are secreted from attached gland cells. Hence, the reservoirs can be considered as closed bio-reactors for producing defensive secretions. We used RNA interference (RNAi) to analyse in vivo functions of proteins in biosynthetic pathways occurring in insect secretions. After a salicyl alcohol oxidase was silenced in juveniles of the poplar leaf beetles, Chrysomela populi, the precursor salicyl alcohol increased to 98 per cent, while salicyl aldehyde was reduced to 2 per cent within 5 days. By analogy, we have silenced a novel protein annotated as a member of the juvenile hormone-binding protein superfamily in the juvenile defensive glands of the related mustard leaf beetle, Phaedon cochleariae. The protein is associated with the cyclization of 8-oxogeranial to iridoids (methylcyclopentanoid monoterpenes) in the larval exudates made clear by the accumulation of the acylic precursor 5 days after RNAi triggering. A similar cyclization reaction produces the secologanin part of indole alkaloids in plants.

Polychlorinated Biphenyl Exposure, Diabetes and Endogenous Hormones: a Cross-sectional Study in Men Previously Employed at a Capacitor Manufacturing Plant

Studies have shown associations of diabetes and endogenous hormones with exposure to a wide variety of organochlorines. We have previously reported positive associations of polychlorinated biphenyls (PCBs) and inverse associations of selected steroid hormones with diabetes in postmenopausal women previously employed in a capacitor manufacturing plant.

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