Increasing uptake and bioactivation with development positively modulate diazinon toxicity in early life stage medaka (Oryzias latipes).

Diazinon, an organophosphate pesticide, becomes biotransformed to a more potent oxon metabolite that inhibits acetylcholinesterase (AChE). Early life stages (els) of medaka, Oryzias latipes, were used to determine how development of this teleost affects sensitivity to diazinon. With developmental progression, from day of fertilization to 7-day-old larvae, we found that the 96-h LC50 and AChE IC50 values decreased, indicating greater host sensitivity to diazinon upon continued development. We then examined changes in AChE activity, its inhibition by the active metabolite diazoxon, and uptake and bioactivation of the compound. AChE activity remained low during much of development but increased rapidly just prior to hatch. In addition, in vitro incubation of tissue homogenates from embryos or larvae showed no differences in the sensitivity of AChE to diazoxon. Uptake studies with 14C-diazinon revealed greater body burdens of 14C as medaka developed. In addition, AChE IC50 values determined by in vivo exposure to diazoxon were greater in larvae than in embryos. Because diazinon is bioactivated by the P450 enzyme system, two P450 inhibitors were used in vivo to explore the role of metabolism in sensitivity. When exposure to diazinon occurred in the presence of increasing amounts of piperonyl butoxide (PBO), AChE inhibition decreased in a dose-response fashion and 2.0 x 10(-5) M PBO alleviated any difference in inhibition between larvae and embryos. However, PBO did not alter total 14C uptake when exposed simultaneously with 14C-diazinon, nor did it affect AChE inhibition using diazoxon. Controls ruled out differential effects of PBO on uptake and inhibition. In addition, a second general P450 inhibitor, 1-aminobenzotriazole, also decreased AChE inhibition. Finally, using exogenous acetylcholinesterase as a trap for the oxon metabolite, larval microsomes displayed greater bioactivation of diazinon than did a microsomal preparation from embryos. Taken together, results suggest that uptake and bioactivation are working to enhance diazinon sensitivity in this developmental model of a teleost fish.

Paternal effects from methamidophos administration in mice.

In this study, the mouse was used to evaluate paternal germline exposure to the organophosphate methamidophos for its potential to produce adverse effects on spermatozoa and in the offspring. There have been reports that organophosphate exposure can increase abnormal sperm morphology in mice. However, effects transmitted to the offspring following paternal exposure have not been reported previously. The maximum tolerated dose (MTD) was 7.5 mg kg(-1) body weight and this dose resulted in no deaths, although blood plasma cholinesterase activity was still decreased. Males were euthanized 4 weeks after an acute intraperitoneal injection of methamidophos (0.5, 3.75, 5.0, and 7.5 mg kg(-1) body wt) and the number of spermatids per gram testes and sperm morphology were analyzed. In this study, abnormal sperm morphology on a per group basis exhibited a dose-response significantly related to increased methamidophos exposure as indicated by regression analysis and a nested ANOVA (p < 0.0001). Preimplantation embryos that were conceived 6 weeks after paternal methamidophos exposure (5 mg kg(-1) body wt) exhibited a significant increase in cleavage arrest. Fertility of males was also affected as shown by a decrease in the number of two- to four-cell embryos per male (postexposure week 6) and an increase in the number of degenerated embryos (postexposure weeks 4-6). We conclude that methamidophos may have the potential to produce transmissible adverse embryonic effects following an acute paternal germline exposure.

Nocturnal 6-hydroxymelatonin sulfate excretion in female workers exposed to magnetic fields.

The objective of this study was to determine whether daytime occupational exposure to extremely low frequency magnetic fields (MFs) suppresses nocturnal melatonin production. Sixty female volunteers were recruited. Thirty-nine worked in a garment factory, and 21 office workers served as a reference group. Exposure assessment was based on the type of sewing machine used and MF measurements around each type of machine. Eye-level MF flux density was used to classify the operators to higher (>1 microT) and lower (0.3-1 microT) exposure categories. A third group of factory workers had diverse MF exposures from other sources. The reference group had average exposure of about 0.15 microT. Urine samples were collected on Friday and Monday for three consecutive weeks. Melatonin production was assessed as urinary 6-hydroxymelatonin sulfate (6-OHMS) excretion. The ratio of Friday morning/Monday morning 6-OHMS was used to test the hypothesis that melatonin production is suppressed after 4 days of occupational MF exposure with significant recovery during the weekend. Possible chronic suppression of melatonin production was evaluated by studying exposure-related differences in the Friday values by multivariate regression analysis. The Monday/Friday ratios were close to 1.0, suggesting that there is no increase in melatonin production over the weekend. The average 6-OHMS excretion on Friday was lower among the factory workers than in the reference group, but no monotonous dose-response was observed. Multivariate regression analysis identified MF exposure, smoking, and age as significant explanatory variables associated with decreased 6-OHMS excretion.

EMF exposure assessment in the Finnish garment industry: evaluation of proposed EMF exposure metrics.

Recently published studies indicate that having worked in occupations that involve moderate to high electromagnetic field (EMF) exposure is a risk factor for neurodegenerative diseases, including Alzheimer's disease. In these studies, the occupational groups most over-represented for EMF exposure comprised seamstresses, dressmakers, and tailors. Future epidemiologic studies designed to evaluate the possibility of a causal relationship between exposure to EMF and a neuro degenerative disease endpoint such as incidence of Alzheimer's disease, will benefit from the measurement of electromagnetic field metrics with potential biological relevance. Data collection methodology in such studies would be highly dependent upon how the metrics are defined. In this research the authors developed and demonstrated (1) protocols for collecting EMF exposure data suitable for estimating a variety of exposure metrics that may have biological relevance, and (2) analytical methods for calculation of these metrics. The authors show how exposure might be estimated under each of the three prominent EMF health-effects mechanism theories and evaluate the assertion that relative exposure ranking is dependent on which mechanism is assumed. The authors also performed AC RMS magnetic flux density measurements, confirming previously reported findings. The results indicate that seamstresses, as an occupational group, should be considered for study of the possible health effects of long-term EMF exposure.

Purine nucleotides modulate proliferation of brown fat preadipocytes.

The hypothesis that purine nucleotides and nucleosides affect brown fat preadipocyte proliferation was tested using isolated rat interscapular brown fat preadipocytes in culture. Daily addition of 100 microM adenosine triphosphate (ATP) (n = 4) to cultures enhanced the relative DNA content by 1.5-fold compared to control cultures (P < 0.05) measured using CyQUANT-GR fluorescence. Higher concentrations of ATP inhibited growth and 500 (n = 2) or 1000 microM ATP (n = 3) almost completely inhibited growth. ATP (100 microM) did not affect while 250-1000 microM ATP decreased protein content relative to control cultures. Adenosine (100 microM; n = 3) did not affect DNA or protein content, but 500 microM and 1000 microM adenosine suppressed brown adipocyte proliferation and inhibited protein synthesis. Cultured brown adipocytes quickly removed or degraded ATP in the culture media as determined by luciferin-luciferase bioluminescence, suggesting that the inhibitory effects of high ATP concentrations may result from its breakdown to adenosine. The results support the conclusion that ATP promotes and adenosine inhibits brown adipocyte proliferation.

Oxime reactivation of RBC acetylcholinesterases for biomonitoring.

A low-variability method to reactivate blood cholinesterases (ChEs) after prior exposure of mammals, including humans, to ChE-inhibiting organophosphate esters (OPs) is presented. A concentration of 10 mM pyridine 2-aldoxime methochloride (2-PAM Cl) was incubated with intact red blood cells (RBCs) and assayed virtually free of interfering oxime and hemoglobin (Hb). Variability was decreased by reducing the number of washing steps and sedimenting RBC ghosts through a 7% sucrose cushion. Statistically significant detections of reactivations as low as 5% with average "false positives" of 3.8% were achieved. Relative rates and extent of reactivation after OP treatment of rabbit RBC AChE in vitro were of the order dimethyl- (DDVP) > diethyl- (ethyl paraoxon) >, diisopropyl-substituted (diisopropyl fluorophosphate; DFP) OPs. Rabbit RBC AChE was reactivatable for up to 60 h following dermal exposure to ethyl parathion and reactivatable for only 12 to 24 h following exposure to methyl parathion. Reactivation of plasma ChEs with 0.1 mM 2-PAM Cl in the same animals was achievable for only 12 to 24 h after ethyl parathion and for only 1 to 4 h after methyl parathion.

Effects of 60 Hz magnetic field exposure on the pineal and hypothalamic-pituitary-gonadal axis in the Siberian hamster (Phodopus sungorus).

Experiments using the dwarf Siberian hamster Phodopus sungorus were carried out to determine possible neuroendocrine consequences of one-time and repeated exposures to 60 Hz magnetic fields (MF). Animals were maintained in either a short-light (SL, 8 h light:16 h dark) or long-light (LL, 16 h light:8 h dark) photoperiod. Acute (one-time, 15 min) exposure of male SL animals to a linearly polarized, horizontally oriented, 60 Hz MF (0.1 mT) gave rise to a statistically significant (P < .005) reduction in pineal melatonin content as determined 3 and 5 h after onset of darkness. In LL animals, acute exposure to 0.10 mT resulted in a significant decrease in pineal melatonin as measured 4 h after onset of darkness, whereas acute exposure to 50 microT showed no effect compared with sham exposure. In SL animals, an increase in norepinephrine was observed in the medial basal hypothalamus (including the suprachiasmatic nucleus) after acute exposure (P < .01). Daily MF exposure of SL animals to a combination of steady-state and on/off 60 Hz magnetic fields (intermittent exposure) at 0.1 mT for 1 h per day for 16 days was associated with a reduction in melatonin concentrations at 4 h after onset of darkness and an increase in blood prolactin concentrations (P < .05). Exposure of SL animals to a steady state 60 Hz MF for 3 h/day for 42 days resulted in a statistically significant reduction in body weight (ANOVA: P > .05), compared with sham-exposed SL animals. At 42 days, however, no significant changes in overnight melatonin or prolactin levels were detected. In both repeated exposure experiments, gonadal weights were lowest in the MF-exposed groups. This difference was statistically significant (P < .05) after 42 days of exposure. These data indicate that both one-time and repeated exposure to a 0.1 mT, 60 Hz MF can give rise to neuroendocrine responses in Phodopus.

Organophosphate-induced acetylcholinesterase inhibition and embryonic retinal cell necrosis in vivo in the teleost (Oryzias latipes).

Recent monitoring of the Sacramento-San Joaquin River system (CA) indicates that levels of the organophosphate pesticide, diazinon, exceed National Academy of Science guidelines and these levels result in toxicity in USEPA acute toxicity tests with Ceriodaphnia dubia. Since organophosphates (OPs) inhibit acetylcholinesterase (AChE), the present study examined the effects of diazinon on the embryonic nervous system of a model teleost, medaka, Oryzias latipes. Preliminary histological screens revealed limited retinal cell necrosis in control embryos with apparent increased necrosis in diazinon-exposed embryos. Subsequently, embryos were exposed to 1.8 x 10(-5), 4.4 x 10(-5), or to 8.8 x 10(-5) M diazinon and replicates were frozen for biochemical analysis or were fixed for histopathological analysis at days 3, 5, and 7 of development. Diazinon exposure significantly inhibited AChE activity within whole embryos and in homogenates of retinas from treated animals. Histological examination of embryos indicated that as the retina underwent differentiation into distinct cell layers, between days 5 and 7, small foci of necrotic cells became apparent within the inner nuclear layer and isolated individual pyknotic cells were observed in the ganglion layer. Quantification of foci of necrotic cells revealed that 8.8 x 10(-5) M diazinon increased number and area of these lesions. Enzyme histochemistry localized AChE activity to regions equivalent to sites of necrosis. Separate exposures of embryos to the OP, diisopropylphosphorofluoridate, produced large foci of necrotic cells at sites equivalent to those seen following diazinon exposure.

Development and validation of a fecal testosterone biomarker in Mus musculus and Peromyscus maniculatus.

This is a report on the development and validation of an ELISA method to determine fecal testosterone levels, and on their evaluation as a biomarker for adverse effects of endocrine-disrupting chemicals on reproductive health using male rodents of the Peromyscus maniculatus and Mus musculus species as an animal model. The ELISA antibody had the highest specificity for testosterone (100%), followed by dihydrotestosterone (57.4%) and androstenediol (0.27%). Radiolabeled testosterone was injected i.p. into three mice. Fecal samples were collected, extracted, and analyzed by liquid scintillation counting. The ELISA was performed to characterize the excretion kinetics and metabolic fate of circulating testosterone. Solubilization of feces with 10% methanol overnight provided an extraction efficiency of 87% for all metabolites; an ethyl ether extraction was more selective for testosterone. The fecal excretion of the testosterone was a biphasic process with a majority of the radioactivity recovered in the first 24 hours. HPLC analysis revealed at least five testosterone metabolites in feces, with most metabolites being less polar than testosterone. This study forms the initial evaluation of what will become a field monitoring tool.

Clinical effects of low-level exposures to chemical warfare agents in mice and chickens.

Concerns that chemical warfare (CW) agents themselves or in combination with other chemicals may cause long-term damage to nerve and muscle are reviewed and discussed. Experiments on mice and hens underway with agent GA and pyridostigmine bromide (PB) and their effects (either separately or together) are presented.

Torsion of the gallbladder: a case report and review of the literature.

Acute torsion of the gallbladder is a rare and poorly understood entity. Since it was first described by A.V. Wendel in 1898, approximately 300 cases have been reported in the literature. The treatment of choice remains immediate cholecystectomy, and most cases are diagnosed intraoperatively. There are typical clinical and radiological findings consistent with torsion of the gallbladder that should raise the index of suspicion for this condition preoperatively. We present a case of gallbladder torsion and discuss the pertinent literature. A re-emphasis is placed on the salient clinical features, and the availability of diagnostic tests is stressed. Given the possibility of laparoscopic cholecystectomy and the increasing incidence with which gallbladder torsion is being witnessed today, the importance of a preoperative suspicion is discussed.

Monitoring the pesticide-exposed worker.

Cholinesterase inhibitors-the organophosphates and the carbamates-are the most acutely toxic and widely used insecticides. They also comprise the only group of pesticides for which state laws exit requiring worker monitoring. This chapter focuses on cholinesterase monitoring, with attention to available assays and testing kits.

Results of a survey to determine whether Neospora is a significant cause of ovine abortion in England and Wales.

Between January and April 1995, the brains and hearts of 281 aborted lambs derived from 209 submissions to veterinary investigation centres in England and Wales were examined histologically. One-hundred-and-seventy-nine samples of fetal pleural fluid from these lambs and 141 from lambs not examined histologically were examined for antibodies to Neospora by an indirect fluorescent antibody test. Non-suppurative myocarditis and encephalitis were present in nine lambs. Immunocytochemistry using antisera against Neospora caninum and Sarcocystis species resulted in no labelling but anti-sera to Toxoplasma gondii revealed labelled organisms in four lambs. No significant antibody titres against Neospora were detected in any of the samples of fetal pleural fluid. These results suggest that Neospora infection is not associated with significant numbers of abortions in sheep in England and Wales.