Environmental endocrine disruption in decapod crustacean larvae: hormone titers, cytochrome P450, and stress protein responses to heptachlor exposure

A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures. Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates. One of the primary functions of stress proteins (HSPs) is to aid in the recovery of damaged proteins by chaperoning their refolding. These and other biomarkers of xenobiotic exposure and resulting effects have not been studied in crustacean larvae. This information is of potential importance for environmental management and risk assessment. In this work, we have given Homarus americanus larvae single 24 h exposures to the cyclodiene pesticide heptachlor, a known environmental endocrine disruptor (EDC) on different days of the 1st larval instar. We followed these larvae during the first larval stage for effects on timing of ecdysis to 2nd stage, ecdysteroid molting hormone titers, and alterations in the levels of cytochrome P450 CYP45 and HSP70 proteins. Delays in ecdysis were correlated with alterations in ecdysteroid levels. This result provides clues that this pesticide may function as an environmental endocrine disruptor in crustaceans. CYP45 and HSP70 levels were significantly elevated for several days following heptachlor exposure. The elevation in HSP70 was prolonged depending on the day of pesticide exposure and this was directly related to the increase in mortality. These results demonstrate the utility of these measurements as potential biomarkers in crustacean larval developmental toxicology and EDC effects research.     

Nephrotoxicity and hepatotoxicity induced by inhaled bromodichloromethane in wild-type and p53-heterozygous mice.

Bromodichloromethane (BDCM) is a common municipal drinking water disinfection by-product, resulting in widespread trace human exposure via ingestion and inhalation. The present studies were designed to define organ-specific, BDCM-induced toxicity in wild type (p53(+/+)) and heterozygous (p53(+/-)) mice on both the FVB/N and C57BL/6 genetic backgrounds. Mice were exposed to BDCM vapor daily for 6 h/day and 7 days/week at concentrations of 0, 1, 10, 30, 100, or 150 ppm for 1 week and at 0, 0.3, 1, 3, 10, or 30 ppm for 3 weeks. In the 1-week exposure study, dose-dependent mortality and morbidity were observed at concentrations of 30 ppm and above and were as high as 100% at 150 ppm. In the 3-week exposure study, mortality and morbidity were found only in the 30-ppm exposure groups and were 0, 17, 67, and 33% for the wild-type C57BL/6, p53(+/-) C57BL/6, wild-type FVB/N, and p53(+/-) FVB/N mice, respectively. BDCM was a particularly potent kidney cytotoxicant. Dose-dependent tubular degeneration, necrosis, and associated regenerative cell proliferation greater than 10-fold over controls were seen at concentrations as low as 10 ppm in the kidneys of all strains at 1 week. Similar dose-dependent increases in hepatic necrosis, degeneration, and regenerative cell proliferation were observed but were induced only at concentrations of 30 ppm and higher. Pathological changes were more severe in the FVB/N compared to the C57BL/6 mice and were more severe in the heterozygotes compared to the wild-type mice. However, recovery and return of the percentage of kidney cells in S-phase to control levels was seen at 3 weeks. The estimated maximum tolerated dose for longer-term exposures was 15 ppm, based on mortality, induced kidney pathology, and regenerative cell proliferation. A one-year cancer bioassay was initiated with doses of 0, 0.5, 3, 10, and 15 ppm, based on this information. No pathological changes in the livers were found at the 13-week time point of that study. At 13 weeks, the kidney lesions and regenerative cell proliferation seen at the 1-week time point at doses of 10 ppm and above had resolved, and the cell proliferation rates had returned to baseline. Differences in toxicity indicate that caution be used in substituting wild-type mice for transgenic mice for range-finding studies to select doses for p53(+/-) cancer studies. Resolution of the kidney lesions indicates that periods of very high regenerative cell proliferation, potentially important in the carcinogenic process, may not be observed if measurements are taken only at 3 weeks of exposure or later.     

No acute toxicity to Uca pugnax, the mud fiddler crab, following a 96-h exposure to sediment-bound permethrin

In coastal areas, the application of pyrethroid insecticides and the resulting sediment residues pose a potential threat to marine benthic ecosystems. Pyrethroids cause acute toxicity and exhibit a wide range of sublethal effects on fish and crustaceans when exposure is aqueous. Fiddler crabs that inhabit salt marsh sediment are sensitive to sediment-associated pollutants and serve as a sentinel species for xenobiotic exposure. We exposed adult U. pugnax to salt marsh sediment spiked with different 60% trans/40% cis permethrin concentrations for 96 h, and evaluated changes in oxygen consumption rate, hemolymph osmolarity, and glutathione S-transferase activity (GST) following exposure. Marsh sediment was not lethal to U. pugnax at permethrin concentrations of 100-10,000 microg/kg. Sediment-bound permethrin had no significant effect on respiration and osmoregulation. Exposure caused an induction of hepatopancreas GST in a dose-dependent manner. Gill and midgut tissues showed induction at permethrin concentrations at 10,000 microg/kg. We conclude that short term exposure to permethrin-contaminated sediment does not pose a significant threat to this species or impact respiration and osmoregulation. Furthermore, increased GST activity allows us to evaluate this enzyme's induction as a generalist biomarker for sediment-bound pyrethroid exposures.     

Early developmental neurotoxicity of a PCB/organochlorine mixture in rodents after gestational and lactational exposure.

The developmental and neurobehavioral effects of gestational and lactational exposure to a mixture of 14 polychlorinated biphenyls (PCBs) and 11 organochlorine pesticides was examined and compared against the commercial PCB mixture Aroclor 1254. The mixture was based on blood levels reported in Canadian populations living in the Great Lakes/St. Lawrence basin. Pregnant Sprague-Dawley rats were dosed orally with 0.013, 0.13, 1.3, or 13 mg/kg of the chemical mixture or 15 mg/kg of Aroclor 1254 from gestation day (GD) 1 to postnatal day (PND) 23. The highest mixture dose decreased maternal gestation and lactation body weight, and produced high mortality rates (80% overall) and reductions in offspring weight that persisted to adulthood. Aroclor 1254 produced smaller but persistent decreases in offspring weight without affecting maternal weight or offspring mortality. Aroclor 1254 and 13 mg/kg of the mixture produced comparable decreases in maternal and offspring serum T4 levels and comparable alterations to maternal thyroid morphology. Aroclor 1254 delayed the righting reflex and ear opening, accelerated eye opening, and reduced grip strength at PNDs 10-14. The mixture at 13 mg/kg delayed negative geotaxis in addition to delaying righting reflex and ear opening and reducing grip strength but had no effect on eye opening. Lower mixture doses (0.13 and 1.3 mg/kg) also delayed ear opening but affected no other parameters. Developmental exposure to the chemical mixture was found to be more toxic than exposure to Aroclor 1254 and produced a different profile of effects on early neurodevelopment. This PCB/organochlorine pesticide mixture affects mortality, growth, thyroid function, and neurobehavioral development in rodents.     

Overall pesticide effects on growth and emergence of two species of Ephemeroptera in a model stream carrying pesticide- polluted river water

The overall pesticide effects on mortality, growth and emergence of two dominant species of Ephemeroptera in Japanese rivers, Epeorus latifolium and Ecdyonurus yoshidae, were assessed using an outdoor channel carrying water from the Kokai River. Young larvae collected from another river were introduced into cages in the channel after their body lengths were measured. The concentrations of 17 pesticides were measured three times a week from April to August 1993. A shrimp mortality test on water samples was conducted concurrently. A relationship between the high mortality of the shrimp in the water samples and of E. latifolium in the channel was recognized. The mortality of E. yoshidae increased only when the shrimp mortality increased drastically in early June, reflecting the difference in insecticide susceptibility between the two mayfly species. Almost all the larvae which had been introduced into the channel in winter and/or early spring, when pesticides had cleared from the river, emerged in spring, although their growth rate during the winter was low. The shrimp mortality in the river water samples was caused by the overall pesticide toxicity. The increase in the mortality of the mayfly larvae in the channel might be due to the overall pesticide toxicity, although their concentrations were low and varied independently     

Comparative toxicity of nickel oxide, nickel sulfate hexahydrate, and nickel subsulfide after 12 days of inhalation exposure to F344/N rats and B6C3F1 mice

The relative toxicity of nickel oxide (NiO), nickel sulfate hexahydrate. (NiSO4.6H2O), and nickel subsulfide (Ni3S2) was studied in F344/N rats and B6C3F1 mice after inhalation exposure for 6 h/day, 5 days/week for 12 exposure days. Exposure concentrations used (as mg Ni/m3) were 0.9-23.6 for NiO; 0.8-13.3 for NiSO4.6H2O, and 0.4-7.3 for Ni3S2. For each compound there were 5 exposure groups plus a control group. NiSO4.6H2O was the most toxic compound with exposure related mortality seen at exposure concentrations of 13.3 mg/m3 in rats and 1.6 mg/m3 and above in mice. For Ni3S2, mortality was seen in mice (but not in rats) at the highest exposure concentration (7.3 mg/m3). No mortality was seen after NiO exposure. Lesions of the lung and nasal cavity were seen in both rats and mice after exposure to NiSO4.6H2O and Ni3S2 at the 4 highest exposure concentrations. Lesions of the lung were seen primarily at the highest exposure concentrations after NiO exposure. The amount of nickel in the lungs at the end of exposure varied in relation to the water solubility of the compounds. Based on these 2-week studies, the toxicity ranking was NiSO4.6H2O greater than Ni3S2 much greater than NiO. Additional studies are in progress to assess the relative toxicities of these three nickel compounds after 90-day exposures.     

Mechanism underlying the effect of long‐term exposure to low dose of pesticides on DNA integrity

Pesticides, including herbicides, insecticides and fungicides, are widely used in intensive agriculture. Recently, the long‐term effects of pesticide exposure were found to be associated with many diseases. In this study, we evaluated the long‐term effect of low‐level exposure to a mixture of pesticides on DNA damage response (DDR) in relation to individual detoxifying variability. A residential population chronically exposed to pesticides was enrolled, biological/environmental pesticide levels; paroxonase 1 (PON‐1) activity and 192 Q/R polymorphism and DDR were evaluated at three different periods of pesticide exposure. OGG1‐dependent DNA repair activity was decreased in relation to pesticide exposure. The increase of DNA lesions and pesticide levels in the intensive pesticide‐spraying period was independent on PON‐1 activity. Next, human bronchial epithelial and neuronal cells were used as a model for in vitro evaluation of the mechanistic effect of pesticides. Pesticides induced mitochondrial dysfunction leading to ROS formation. ROS from mitochondria induced DNA damage, which in turn induced OGG1‐dependent DNA repair activity through 8‐oxoguanine DNA glycosylase 1 (OGG1) expression and activation. Even though OGG1 was overexpressed, an inhibition of its activity, associated with DNA lesion accumulation, was found at prolonged pesticide‐exposure. A post‐translational regulation of OGG1 by pesticide may be postulated. Taken together, long‐term exposure to low‐levels of pesticides affects DDR resulting in accumulation of DNA lesions that eventually may lead to cancer or neurological disorders.     

Inhibition of molting by cadmium in the crab Chasmagnathus granulata (Decapoda Brachyura)

The effect of cadmium on molting of the estuarine crab Chasmagnathus granulata was assayed. Adult males were eyestalk-ablated, in order to synchronically induce molting, and were then chronically exposed to cadmium at 0.25 or 0.50 mg/l. At the highest concentration, a significant mortality was detected at the time of molting, in the few crabs that could reach the E stage. However, most of the crabs exposed from the beginning of the premolt period (D(0) stage) to 0.50 mg/l of cadmium were arrested at the D(1)" stage. This effect was not seen when crabs were exposed to the same cadmium concentration from either D(1)"' or D(3) premolt stages. Crabs arrested by cadmium did not present any difference in the calcium content of carapace, compared to controls, while ecdysteroid levels of those crabs were similar to the ones of control crabs that were in the same premolt stage but could finally molt. These results suggest that cadmium could be preventing the normal peaking of ecdysteroids needed for molting. Since eyestalk-ablated crabs were used, a presumably direct effect of cadmium on Y-organ seems likely, by affecting cytoplasmatic calcium concentration and/or other actions.     

Effects of ethylene dibromide on reproduction in male and female rats

The effects of ethylene dibromide (EDB) on reproduction were studied in both sexes of rats. Males inhaled average daily concentrations of 19, 39, and 89 ppm of EDB 7hr/day, 5 days/week, for 10 weeks. Mortality and morbidity occurred in the group exposed to the high concentration. In addition, males in this group had reduced testicular weights, reduced serum testosterone concentrations, and failed to impregnate any females during a 2-week mating period. Atrophy of the testes, epididymis, prostate, and seminal vesicles was observed in these males. The reproductive performance of males exposed to 19 or 39 ppm of EDB was not impaired. Females inhaled average daily concentrations of 20, 39, or 80 ppm of EDB 7 hr/day, 7 days/week, for 3 weeks. Mortality and morbidity occurred in the group exposed to the high concentration. Females in this group did not cycle normally until several days after the termination of exposure. The reproductive performance of females exposed to 20 or 39 ppm of EDB was normal. Pregnant females in all groups produced normal litters, and histopathological examination of the ovary and uterus did not reveal lesions that would impair reproductive performance. Thus, adverse effects on reproduction were observed in both sexes of rats only at concentrations of EDB that also produced mortality and morbidity. Therefore, it was not possible to attribute the reproductive effects directly to EDB.     

Evaluating acute toxicity of methyl parathion application in constructed wetland mesocosms

Wetland ecosystems have reduced ambient levels of various organic and metallic compounds, although their effectiveness on agricultural pesticides is not well documented. Five stations within each of two 10 x 50 m constructed wetlands (two vegetated, two nonvegetated) were selected to measure the fate and effects of methyl parathion (MeP). Following a simulated storm event (0.64 cm of rainfall), aqueous, sediment, and plant samples were collected and analyzed spatially (5, 10, 20, and 40 m from the inlet) and temporally (after 3-10 days) for MeP concentrations and for the impact of those concentrations on the aquatic fauna. Aqueous toxicity to fish decreased spatially and temporally in the vegetated mesocosm. Pimephales promelas survival was significantly reduced, to 68%, at the 10-m station of the nonvegetated wetlands (3 h postapplication), with pesticide concentrations averaging 9.6 microg MeP/L. Ceriodaphnia in both the vegetated and nonvegetated wetlands was sensitive (i.e., a significant acute response to MeP occurred) to pesticide concentrations through 10 days postapplication. Mean MeP concentrations in water ranged from 0.5 to 15.4 microg/L and from 0.1 to 27.0 microg/L in the vegetated and nonvegetated wetlands, respectively. Hyalella azteca aqueous tests resulted in significant mortality in the 5-m vegetated segment 10 days after exposure to MeP (2.2 microg/L). Solid-phase (10-day) sediment toxicity tests showed no significant reduction in Chironomus tentans survival or growth, except for the sediments sampled 3 h postapplication in the nonvegetated wetland (65% survival). Thereafter, midge survival averaged >87% in sediments sampled from both wetlands. These data suggest that wetlands play a significant role in mitigating the effect of MeP exposure in sensitive aquatic biota.     

Toxicity of aldicarb in young chicks

Enzyme and locomotion changes due to carbamate treatment were investigated during and following oral administration of aldicarb (0.2 mg/kg body weight/day ×7) to six-day-old chicks. Brain acetylcholinesterase (AChE) and neurotoxic esterase (NTE) were measured at three times during the one week of treatment, and again on days 1, 3, 6, 10, 20, 30 and 40 after the last dose. Gait analysis was evaluated on each posttreatment day at which the biochemical assays were performed. Multiple doses of aldicarb resulted in a significant reduction in the weight of the treated chicks beginning 6 days after the last treatment. Treatment with aldicarb had no significant effect on brain NTE during or after dosing. The AChE levels in treated chicks were significantly lower than those of controls only 24 hr after the first dose. Significant alterations in the locomotion of the chicks was seen from day 1 until day 40 after last treatment. Treated chicks had significantly shorter stride length, longer stride width and wider sine of the angle of placement than the controls. This study demonstrated that repeated exposure to aldicarb altered locomotion of the chicks for several weeks after cessation of exposure with no significant esterases inhibition, indicating that behavioral alterations due to pesticide exposure need not be associated with enzyme changes.     

A 90-Day Chloroform Inhalation Study in Female and Male B6C3F1 Mice: Implications for Cancer Risk Assessment

High doses of chloroform induced liver cancer in male and femaleB6C3F₁ mice when administered by gavage, kidney cancer in maleOsborne-Mendel rats when given by gavage or in the drinkingwater, and kidney cancer in male BDF₁ mice when administeredby inhalation. The weight of evidence indicates that chloroformis acting through a nongenotoxic-cytotoxic mode of action. Thepresent study was designed to investigate the dose-responserelationships for chloroform-induced lesions and regenerativecell proliferation in B6C3F₁ mice as the basis for formulationof a biologically based risk assessment for inhaled chloroform.Different groups of female and male B6C3F₁ mice were exposedto atmospheric concentrations of 0, 0.3, 2, 10, 30, and 90 ppmchloroform 6 hr/day, 7 days/week for exposure periods of 4 daysor 3, 6, or 13 consecutive weeks. Some additional exposure groupswere exposed for 5 days/week for 13 weeks or were exposed for6 weeks and then examined at 13 weeks. Bromodeoxyuridine wasadministered via osmotic pumps implanted 3.5 days prior to necropsy,and the labeling index (LI, percentage of nuclei in S-phase)was evaluated iminunohistochemically from histological sections.Complete necropsy and microscopic evaluation revealed treatment-induceddose- and time-dependent lesions only in the livers and nasalpassages of the female and male mice and in the kidneys of themale mice. Large, sustained increases in the liver LI were seenin the 90-ppm groups at all time points. The female mice weremost sensitive, with a no-observed-adverse-effect level (NOAEL)for induced hepatic cell proliferation of 10 ppm. The hepaticLI in the 5 days/week groups were about half of those seen inthe 7 days/week groups and had returned to the normal baselinein the 6-week recovery groups. Induced renal histologic changesand regenerative cell proliferation were seen in the male miceat 30 and 90 ppm with 7 days/week exposures and also at 10 ppmwith the 5 days/week regimen. Nasal lesions were transient andconfined to mice exposed to 10, 30, or 90 ppm for 4 days. Ina previous cancer bioassay, a gavage dose of 477 mg/kg/day produced a 95% liver tumor incidence in female B6C3F₁ mice. Thisgavage dose is equivalent to a daily 6 hr/day inhalation exposureof approximately 80 ppm, based on the observed induced increasesin the LI as an internal dosimeter. The United States EnviromnentalProtection Agency currently uses the linearized multistage modelapplied to the mouse liver tumor data from the chloroform gavagestudy to estimate a virtually safe dose (VSD) as a one in amillion increased lifetime risk of cancer. The resulting valueis an airborne exposure concentration of 0.000008 ppm. Assumingthat chloroform-induced female mouse liver cancer is secondaryto events associated with necrosis and regenerative cell proliferation,then no increases in liver cancer in female mice would be predictedat the NOAEL of 10 ppm or below based on the results reportedhere. Applying an uncertainty factor of 1000 yields an estimateof a VSD at 0.01 ppm. This estimate relies on inhalation dataand is more consistent with the mode of action of chloroform.     

Tadpole size, cholinesterase activity, and swim speed in four frog species after exposure to sub-lethal concentrations of chlorpyrifos

While physiological biomarkers exist to verify exposure of amphibians in natural populations to agricultural chemicals, the ecological relevance of changes in these parameters is often difficult to determine. We compare the relationship between tadpole cholinesterase (ChE; a common enzymatic biomarker of exposure to OP pesticides) and measures of size and swim speed in four native North American species of anurans (Hyla chrysoscelis, Rana sphenocephala, Acris crepitans, and Gastrophryne olivacea). We used four environmentally realistic levels (1, 10, 100 and 200 μg/l) of a commonly used organophosphate pesticide (OP), chlorpyrifos, and examined tadpole response at the conclusion of 4 days of exposure. We further examined if the presence or absence of pond sediment influenced tadpole responses, and, in two species, we determined how a 12-day exposure influenced responses. We found species-specific differences in response to the pesticide, with H. chrsysoscelis and G. olivacea being most sensitive; however, the levels of inhibition of ChE activity were generally not sufficient to exert an effect on swim speed as we measured it. Generally, tadpole mass was reduced 20–35% in the highest concentration after 4 days of exposure. We found the presence of sediment to influence these responses, although the effects were not consistent among species. Given these differences in ecologically relevant responses, we recommend exercising caution when making generalizations across different anuran amphibian taxa regarding responses to pesticide exposure.     

The joint neurotoxic action of inhaled methyl butyl ketone vapor and dermally applied O-ethyl O-4-nitrophenyl phenylphosphonothioate in hens: potentiating effect

The neurotoxic action of inhaled technical grade methyl butyl ketone and dermally applied (O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was studied. Three groups of five hens each were treated 5 days/week for 90 days with a dermal dose of 1.0 mg/kg of EPN (85%) on the unprotected back of the neck. These groups were exposed simultaneously to 10, 50, or 100 ppm of technical methyl butyl ketone (MBK; methyl n-butyl ketone:methyl isobutyl ketone, 7:3) in inhalation chambers. A fourth group was treated only with the dose of EPN and a fifth group with only 100 ppm MBK. The control consisted of a group of five hens treated with a dose of 0.1 ml acetone. Treatment was followed by a 30-day observation period. Simultaneous exposure to EPN and MBK greatly enhanced the neurotoxicity produced when compared to the neurotoxicity produced by either chemical when applied alone. Continued exposure to EPN and MBK resulted in earlier onset and more severe signs of neurotoxicity than exposure to either individual compound. The severity and characteristics of histopathologic lesions in hens given the same daily dermal dose of EPN in combination with inhaled MBK depended on the MBK concentration. Histopathologic changes were more severe and prevalent in the 100 ppm MBK:1 mg/kg EPN group than in the others. In this group, Wallerian-type degeneration was seen along with paranodal axonal swellings. The morphology and distribution of these lesions were characteristic of those induced by MBK. In the 50 ppm MBK:1 mg/kg EPN group axonal swelling was evident but not clearly identifiable as paranodal. Hens treated with 10 ppm MBK:1 mg/kg EPN had minimal lesions with low incidence of axonal swellings. These were not as large as those seen in MBK neurotoxicity, but instead resembled the histopathologic lesions caused by EPN. The results indicate that the combined treatment gave a value for neurotoxicity coefficient which was two times the additive neurotoxic effect of each treatment alone. Pretreatment with three daily ip doses of 5 mmol/kg technical grade MBK or methyl n-butyl ketone (MnBK), equally increased chicken hepatic microsomal cytochrome P-450 content. Also, hepatic microsomes from MBK-treated hens metabolized [14C]EPN in vitro to [14C]EPN oxon to a much greater extent than those from control hens. These results suggest that MBK potentiates the neurotoxic effect of EPN, at least in part, by increasing the metabolic activation of EPN to the more neurotoxic metabolite EPN oxon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of hepatocellular resistance and susceptibility to styrene toxicity in B6C3F1 mice.

Short-term inhalation exposure of B6C3F1 mice to styrene causes necrosis of centrilobular (CL) hepatocytes. However, in spite of continued exposure, the necrotic parenchyma is rapidly regenerated, indicating resistance by regenerated cells to styrene toxicity. These studies were conducted to test the hypothesis that resistance to repeated styrene exposure is due to sustained cell proliferation, with production of hepatocytes that have reduced metabolic capacity. Male mice were exposed to air or 500 ppm styrene (6 h/day); hepatotoxicity was evaluated by microscopic examination, serum liver enzyme levels, and bromodeoxyuridine (BrdU)-labeling index (LI). Metabolism was assessed by measurement of blood styrene and styrene oxide. Both single and repeated exposures to styrene resulted in mortality by Day 2; in mice that survived, there was CL necrosis with elevated BrdU LI at Day 6, and complete restoration of the necrotic parenchyma by Day 15. The BrdU LI in mice given a single exposure had returned to control levels by Day 15. Re-exposure of these mice on Day 15 resulted in additional mortality and hepatocellular necrosis, indicating that regenerated CL cells were again susceptible to the cytolethal effect of styrene following a 14-day recovery. However, in mice repeatedly exposed to styrene for 14 days, the BrdU LI remained significantly increased on Day 15, with preferential labeling of CL hepatocytes with enlarged nuclei (karyomegaly). If repeated exposures were followed by a 10-day recovery period, CL karyomegaly persisted, but the BrdU LI returned to control level and CL hepatocytes became susceptible again to styrene toxicity as demonstrated by additional mortality and acute necrosis after a challenge exposure. These findings indicated a requirement for continued styrene exposure and DNA synthesis in order to maintain this resistant phenotype. Analyses of proliferating-cell nuclear-antigen (PCNA) labeling were conducted to further characterize the cell cycle kinetics of these hepatocytes. The proportion of cells in S-phase was increased by repeated exposure. However, PCNA analysis also revealed an even larger increase in the G1 cell compartment with repeated exposures, without a concurrent increase in G2 phase or in mitotic cell numbers. These data indicate that resistance to styrene-induced necrosis under conditions of repeated exposure is not due to sustained cell turnover and production of new, metabolically inactive cells, but rather is due to some other, as yet unknown, protective phenotype of the regenerated cells.     

An OGG1 polymorphism is associated with mitochondrial DNA content in pesticide-exposed fruit growers

Exposure to pesticides has the capacity to cause mitochondrial dysfunction. An increase mitochondrial DNA (mtDNA) content has also been suggested to relate with DNA damaging agent. In mitochondria, the manganese superoxide dismutase (MnSOD) is a scavenger of reactive oxygen species, and the 8-oxoguanine DNA glycosylase (OGG1) is the major DNA glycosylase for the repair of 8-oxoG lesions. However, the alteration of mtDNA content elicited by pesticide exposure in people with genetic variations in MnSOD or OGG1 has not been investigated. In this study, the mitochondrial to nuclear DNA ratio was quantified in the peripheral blood of 120 fruit growers who experienced pesticide exposure and 106 unexposed controls by real-time quantitative polymerase chain reaction (real-time qPCR). Questionnaires were administered to obtain demographic data and occupational history. The MnSOD and OGG1 genotypes were identified by the PCR based restriction fragment length polymorphism analysis. After adjusting for confounding effects, multiple regression model revealed that subjects experiencing high or low pesticide exposure had a greater mtDNA content than that of controls. The OGG1 Ser-Ser genotype was also associated with an increased mtDNA content. No association between MnSOD genotype and mtDNA content was revealed. Thus, subjects experiencing pesticide exposure had greater mtDNA content and the OGG1 genotype may modulate mtDNA content in pesticide-exposed fruit growers.     

Tumors of the Skin in the HRA/Skh Mouse after Treatment with 8-Methoxypsoralen and UVA Radiation

8-Methoxypsoralen (8-MOP) with and without UVA radiation wasadministered to HRA/Skh mice (36 animals per treatment group)three times a week in the feed for a total dose of 9–80mg/kg/week for 52 weeks. Most of the animals at the top doseof 8-MOP with UVA radiation had developed skin toxicity and/orskin tumors by 52 weeks. The skin lesions seen after treatmentwith 8-MOP and UVA radiation were characterized as squamouscell hyperplasia, squamous cell papilloma, and squamous cellcarcinoma and are similar to what has been reported in humansafter exposure to 8-MOP and UVA. Squamous cell hyperplasia andacute inflammation of the cornea were also seen in some of thetreated female mice. Oral administration of 8-MOP and UVA didnot result in a carcinogenic response to other organ systems.There were no increases in skin neoplasms after 8-MOP or UVAradiation alone. 8-MOP given in combination with UVA was carcinogenicto the skin of mice at dose levels similar to those used totreat psoriasis in humans.     

Selective down-regulation of D1 dopamine mediated rotational behavior in supersensitive mice

The effects of continuous exposure to selective D1 and D2 agonists on rotational behavior were examined in mice with unilateral 6-hydroxydopamine-induced lesions of the corpus striatum. Continuously exposing mice to the D1 agonist SKF 38393 produced an initial rotational behavioral response which decreased dramatically within 4 hours. At the time these rotations ceased, the mice were totally unresponsive to an acute challenge injection of SKF 38393 but responded normally to an acute injection of the D2 agonist quinpirole. Mice chronically implanted with quinpirole also showed marked rotational behavior, but in contrast to the results with SKF 38393, essentially no diminution of these rotations was seen during a one week period of exposure. The results suggest that there are different mechanisms involved in the long term regulation of D1 and D2 dopamine systems in supersensitive animals.     

Influence of genetic composition of test-animal populations on chronic toxicity studies used for risk estimation

A lifespan exposure of mice to benzidine dihydrochloride was conducted for 33 m using both sexes of two populations of mice with the same gene pool. One population was the genetically homogeneous F1 hybrid produced by crossing BALB/cStCrlC3Hf/Nctr males with C57BL/6jfC3Hf/Nctr females. The second population consisted of genetically heterogenous monohybrid cross (MC) offspring produced by mating the F1 hybrids inter se. Data comparisons were made to determine if gene distribution among members of a population affects the response to a toxic insult. Endpoints tested consisted of mortality, liver tumor incidence and time of tumor onset, mortality from reticulum-cell sarcoma, and body weights. In most instances it was noted that among animals not dosed (controls), the F1 population had lower background incidence of lesions and lived longer than the MC population. However, among the dosed animals, the F1 mice were generally more susceptible to the toxic agent and developed higher incidences of the chemically induced lesions than did the MC population. The F1 hybrid population gave a more conservative estimate of risk than did the MC population. The calculation of the liver tumor risk for these two populations showed that lifespan exposure to benzidine would be predicted to result in a larger number (higher risk) when using the F1 data. A 4.5-fold difference in the toxic response was observed between the F1 females and the MC males. This emphasizes the importance of gene distribution in risk estimation studies.     

Ecological relevance and sensitivity depending on the exposure time for two biomarkers

Biomarkers are widely used to assess pesticide stress, but their ecological relevance and exposure time dependent sensitivity is still heavily debated. We studied both aspects in larvae of the damselfly Coenagrion puella, comparing the impact of low doses of atrazine, carbaryl, and endosulfan on two key biomarkers (acetylcholinesterase [AChE] activity and fluctuating asymmetry [FA]) and their relationship with life history traits (mortality, development time, growth rate, and body size). Larvae exposed to the pesticides had, in general, longer development times. Size, growth rate, and mortality were not affected by any of the pesticides. In the long-term exposure, AChE activity was diminished by atrazine treatments and stimulated by carbaryl treatments, and was not affected in the endosulfan treatments. FA decreased with increasing endosulfan concentrations and showed no reaction to atrazine or carbaryl. Overall, short-term exposure tended to overestimate the results of long-term exposure decreasing growth rates and enhancing inhibition of AChE activity in atrazine and carbaryl treatments. In line with its ecological relevance, relationship between biomarkers and life history traits showed that AChE inhibition was positively correlated with mortality, while FA was traded off with size. These results show that caution should be exerted when using these biomarkers to assess pesticide pollution in field situations.