Activities of liver and lung cytochrome P450-dependent monooxygenases and antioxidant enzymes in laboratory and wild Norway rats exposed to reference and contaminated soils

Laboratory and wild Norway rats were exposed in the laboratory to an uncontaminated soil and to a soil from a site contaminated with petrochemical waste. Activities of microsomal lung and liver cytochrome P450-dependent monooxygenases, including 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD) and 7-benzoxyresorufin O-debenzylase (BROD) were measured at selected times during the course of the study. The highest degree of induction of hepatic EROD (7-fold) was shown after 3 days of exposure to the contaminated soil. However, two months later, the EROD activity declined to fourfold increase over the control. The PROD and BROD activities displayed a similar time course of induction, but the degree of induction was lower. The induction of hepatic monooxygenase activities was observed in both laboratory and wild rats. Lung monooxygenase EROD was highly induced (up to 28-fold) after 3 days of exposure, and the activity remained elevated throughout the two-month experiment. BROD and PROD activities were not induced. The activities of three antioxidant enzymes, namely superoxide dismutase, glutathione peroxidase (Se- and non-Se-dependent) and catalase also were measured in lung and liver cytosol, but no significant changes were observed after two months of exposure to contaminated soil.     

Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction

We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.     

Liver mixed function oxidases in chickens: Induction by polychlorinated biphenyls and lack of induction by DDT

Microsomal hydroxylase and demethylase activities were compared in livers from White Leghorn cockerels fed polychlorinated biphenyls (PCBs) containing 21 to 68% chlorine, orp,p′-DDT. The higher chlorinated PCBs, Aroclor 1254 and 1268, were potent inducers of hepatic enzyme activity while DDT did not induce these enzymes. Aroclor 1254 was the most potent inducer because feeding 5 ppm increased liver aminopyrine demethylase activity 1.5X but did not affect liver weight. Feeding 50 and 500 ppm of Aroclor 1254 and 1268 increased liver weight and demethylase activity per mg of protein. A differential response in the two enzymes occurred. Only 500 ppm of Aroclor 1268 increased hydroxylase activity. Aroclor 1242 had no effect on liver enzyme concentration but 50 and 500 ppm of Aroclor 1242 increased liver weights and consequently increased total enzyme activity. Our data demonstrated that in the avian species DDT and PCB did not have the same effect on hepatic microsomal enzyme activity.     

Liver mixed function oxidases in chickens: induction by polychlorinated biphenyls and lack of induction by DDT.

Microsomal hydroxylase and demethylase activities were compared in livers from White Leghorn cockerels fed polychlorinated biphenyls (PCBs) containing 21 to 68% chlorine, or p,p'-DDT. The higher chlorinated PCBs, Aroclor 1254 and 1268, were potent inducers of hepatic enzyme activity while DDT did not induce these enzymes. Aroclor 1254 was the most potent inducer because feeding 5 ppm increased liver aminopyrine demethylase activity 1.5X but did not affect liver weight. Feeding 50 and 500 ppm of Aroclor 1254 and 1268 increased liver weight and demethylase activity per mg of protein. A differential response in the two enzymes occurred. Only 500 ppm of Aroclor 1268 increased hydroxylase activity. Aroclor 1242 had no effect on liver enzyme concentration but 50 and 500 ppm of Aroclor 1242 increased liver weights and consequently increased total enzyme activity. Our data demonstrated that in the avian species DDT and PCB did not have the same effect on hepatic microsomal enzyme activity.     

Environmental polychlorinated biphenyl exposure and cytochromes P450 in raccoons (Procyon lotor)

An investigation involving raccoons as a sentinel species at the Paducah Gaseous Diffusion Plant (PGDP) and Ballard Wildlife Management Area in western Kentucky (USA) delineated the extent of exposure to polychlorinated biphenyls (PCBs). Three separate measures of hepatic cytochrome P450 (CYP) induction were used to evaluate raccoon physiological responses to PCB exposure. Hepatic CYP induction was estimated via determination of total CYP, dealkylase activities, and immunoreactive proteins. There were no differences in raccoon biomarker responses between study sites. Significant relationships between and among PCB residues and biomarkers indicated that hepatic CYP induction had occurred in response to PCB exposure. Pentoxyresorufin O-deethylase (PROD) activity, CYP1A1, and CYP1A2 were biomarkers most closely associated with PCB exposure. The rank order of responses was CYP1A1 > CYP1A2 > PROD > ethoxyresorufin O-deethylase (EROD) as related to raccoon liver PCB concentrations, whereas the order was CYP1A1 > PROD > EROD > CYP1A2 when regressed with total PCB concentrations in abdominal fat.     

Effects of polychlorinated biphenyls on thyroid hormones and liver type I monodeiodinase in the chick embryo.

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants which can biomagnify to higher tropic level organisms including birds. Circulating thyroid hormones (TH) and growth are decreased by PCB exposure. The first set of studies investigated the effects of PCBs on an enzyme responsible for TH homeostasis, hepatic type I monodeiodinase (MDI) in chicken embryos. Fertile chicken eggs were injected with Aroclor 1242, Aroclor 1254, 2,2',6, 6'-tetrachlorobiphenyl (TCB), 3,3',4,4'-TCB, or 3,3',5,5'-TCB on Day 0 and studies were terminated on Incubation Day 21. Hepatic MDI activity was reduced in embryos treated with the Aroclor mixtures. No effects on MDI activities were observed after PCB isomer treatment. Liver weights from embryos treated with Aroclor 1242 were decreased. In the second study, chick embryos were exposed to these same PCBs in order to evaluate their effect on circulating THs and growth. Treatment with PCBs had no effect on body weight. Femur length were decreased with Arcolor 1242 treatment. A decrease in plasma concentration of thyroxine was observed after treatment with Aroclor 1242 and Aroclor 1254. Based on these findings, it is evident that PCBs alter the thyroid axis. Bird circulating TH levels, which are generally reported, may not be a good biomarker for low-dose exposure to PCBs. However, the reduction in MDI activity was more sensitive to PCB mixture exposure and may be a useful biomarker.     

European starling (Sturnus vulgaris): avian model and monitor of polychlorinated biphenyl contamination at a Superfund site in southern Illinois, USA

Accumulation and effects of polychlorinated biphenyls (PCBs) in avian species were evaluated at a Superfund site located at Crab Orchard National Wildlife Refuge (IL, USA). European starlings (Sturnus vulgaris) were monitored at nest boxes constructed at each of two study and two reference sites. During the breeding season, starling productivity and adult provisioning behavior were monitored. At 15 d after hatch, chicks and adults were collected for contaminant and biomarker analyses. Chicks and adults were necropsied. ethoxyresorufin-O-deethylase (EROD) activity was measured in liver tissue, and polychlorinatedbiphenyl (PCB; Aroclor 1254) and 34 chlorinated biphenyl (CB) congener concentrations were measured in carcasses. Polychlorinated biphenyl and CB concentrations also were measured in eggs that failed to hatch. Mean Aroclor 1254 and quantified CB concentrations were greater (p < 0.001) in eggs that failed to hatch, 15-d-old chicks, and adults collected from PCB-contaminated sites (hereafter PCB sites) compared to those collected from reference sites. The EROD activity was greater (p = 0.005) in 15-d-old chicks, but not adults (p = 0.972), collected from PCB sites compared to those collected from reference sites. No differences were found among study and reference sites in number of eggs laid or percent of eggs hatched; however, reduced nest provisioning behavior and decreased chick survival were observed at PCB sites. Polychlorinated biphenyl concentrations measured in samples collected from the PCB sites are similar to concentrations previously associated with adverse effects in avian species. Other avian species utilizing these sites also may be exposed to PCBs with associated adverse effects. This study demonstrates that starling nestlings are good biological monitors of local contamination and continued monitoring may provide useful data for evaluating the effectiveness of proposed remediation.     

Biological Effects of Gestational and Lactational PCB Exposure in Neonatal and Juvenile C57BL/6 Mice

The purpose of this study was to obtain a better understanding of polychlorinated biphenyl (PCB) immunotoxicity in the developing mouse. Adult female mice were dosed with three subcutaneous injections per week of 50 mg/kg Aroclor 1242 (A1242), Aroclor 1254 (A1254), or corn oil for 2 weeks and then mated with nondosed males. First-litter pups were sacrificed at 7 or 28 days of age. At both ages, the tissue concentration of PCB was significantly higher in both the A1242 and A1254 pups than in oil-treated controls. Seven-day-old pups exposed to A1242 or A1254 had significantly decreased splenic IL-2 production. Alterations in the percentages of T cell subsets compared to controls were observed in A1242-exposed pups; an increased spleen somatic index was noted only in A1254-exposed pups. Twenty-eight-day-old pups exposed to A1254 demonstrated a significant decrease in thymus somatic index, an increase in liver somatic index, a 25% decrease in total circulating T₄, and decreased B cell percentages relative to their controls. Alteration in the percentages of CD3^int T cells was observed in A1254-exposed 28-day-old pups. A significant increase in 7-ethoxyresorufin-O-deethylase (EROD) and 7-benzoxyresorufin-O-dearylase (BROD) activity was measured at both ages in A1254-exposed pups and in A1242-exposed 28-day-old pups. These data confirm that during gestation and lactation A1242 and A1254 are transferred from dams to pups and that such exposure results in immune-related effects in neonatal (7-day-old) and juvenile (28-day-old) mice. Furthermore, A1254 exposure produces more frequent and pronounced effects than exposure to A1242. Received: 11 February 2002/Accepted: 2 June 2002     

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.     

Neurobehavioral and somatic effects of perinatal PCB exposure in rats

Developing rats were exposed to PCBs via provision of diets containing 0.02 (no PCB added), 2.5, 26, or 269 ppm Aroclor 1254 to sperm-positive female rats from mating to weaning of their pups. Provision of the 269 ppm diet decreased the number of impregnated rats that delivered a litter and lowered pup birth weight, and most pups died within 7 days of birth. Pregnancy success, pup birth weight, and dam body weight and food intake were not altered in the 2.5 and 26 ppm conditions. Preweaning pup growth was reduced in the 26 ppm condition and slightly reduced in the 2.5 ppm condition. The ontogeny of negative geotaxis, auditory startle, and air righting was delayed in pups from the 26 ppm condition. Pups in the 2.5 ppm condition had slightly delayed development of auditory startle. PCB exposure did not affect the duration of forepaw suspension or age at eye opening. Maximal electroshock seizure tests on postweaning rats showed that perinatal PCB exposure decreased seizure severity of both the 2.5 and 26 ppm groups as indicated by increased durations of forelimb and hindlimb flexion and decreased duration of hindlimb extension. PCB exposure increased pup liver weights at birth and dam and pup liver weights at weaning. Spleen and thymus weights were lower in PCB-exposed pups, while brain weights were unaffected. Analytical determination of PCB levels in brain showed greater maternal transfer of PCBs during lactation than during gestation. Elevated PCB levels were detectable in brains of perinatally exposed adult rats. The results indicate that perinatal PCB exposure of rats alters neurobehavioral and somatic ontogeny.     

Differential effects of two lots of aroclor 1254 on enzyme induction, thyroid hormones, and oxidative stress.

Aroclor 1254 is a commercial mixture of polychlorinated biphenyls (PCBs), which is defined as being 54% chlorine by weight. However, the congener composition varies from lot to lot. Two lots which have been used in toxicity studies, 124-191 and 6024 (AccuStandard), were analyzed for their congener composition. Lot 6024 has approximately 10 times the dioxin toxic equivalents (TEQ) of lot 124-191. The purpose of this study was to determine if the difference in the TEQ of the two lots explains the different in vivo responses seen on a weight basis. Male Long-Evans rats (70 days old) were treated orally with a single dose of 0-1,000 mg/kg of each lot. Hepatic ethoxy-, methoxy-, and pentoxyresorufin O-deethylase (EROD, MROD, and PROD, respectively) activities as well as serum thyroxine (T(4)) concentrations and measures of oxidative stress were determined 4 days after treatment. Results, on a weight basis, indicate that lot 6024 led to a greater induction of EROD, MROD, and PROD but not total T(4) reduction. The differences in TEQ between the lots explained the differential induction of EROD and MROD but did not account for the induction of PROD nor decreases in T(4). PROD induction is not due to dioxin-like congeners, whereas the decrease in serum T(4) levels may involve multiple mechanisms. Effects on the antioxidants ascorbic acid and uric acid were seen only at the highest mass dose for both lots and were not explained by the difference in TEQ. These results illustrate that the differences in the TEQ explain the differences in the strict dioxin-like effects (EROD, MROD induction), but the non-dioxin-like congeners cause other effects that are not associated with the aryl hydrocarbon receptor (e.g., PROD). In addition, supra-additive effects also occur in the mixture (T(4), oxidative stress). Thus, current results demonstrate that overall toxicity cannot be predicted on the basis of the TEQ values. It is also critical that the lot number is reported in studies conducted with Aroclor 1254 because the congener composition and therefore the effects observed can be very different.     

Lipopolysaccharide-induced hepatic injury is enhanced by polychlorinated biphenyls.

After intravenous administration of bacterial lipopolysaccharide (LPS) to rats, polymorphonuclear neutrophils (PMNs) rapidly accumulate in the liver, and midzonal hepatic necrosis is prominent by 6 hr. PMNs are required for the development of hepatic injury in rats. Certain polychlorinated biphenyls (PCBs) can activate PMNs, resulting in production of superoxide anion (O2-.) and release of cytolytic factors from granules. This raises the possibility that PCB exposure might enhance PMN-mediated tissue injury, such as LPS-induced hepatotoxicity. We treated female Sprague-Dawley rats with a minimally toxic dose of LPS in saline (2 mg/kg, intravenous) and 90 min later exposed them to Aroclor 1248 (50 mg/kg, intraperitoneal), a mixture of PCBs. The animals were killed 6 hr after LPS administration, and hepatic injury was assessed. Neither LPS nor Aroclor 1248 alone produced liver injury. Co-treatment with LPS and Aroclor 1248 resulted in pronounced liver injury as demonstrated from increased activities of alanine aminotransferase and isocitrate dehydrogenase in plasma. Histological evaluation indicated increased severity of hepatic necrosis in rats receiving both LPS and Aroclor 1248. Hepatic accumulation of PMNs, normally observed after LPS, was not altered by co-exposure to PCBs. Aroclor 1248 stimulated rat PMNs in vitro to produce O2-. and to degranulate. In addition, PMN-mediated cytotoxicity to isolated rat hepatocytes in culture was increased upon addition of Aroclor 1248. PCBs activate PMNs in vitro and increase PMN-dependent hepatocellular damage in vitro and after LPS treatment in vivo. PCBs may act in vivo as an additional inflammatory stimulus to activate PMNs to become cytotoxic, resulting in increased tissue injury.     

The effects of polychlorinated biphenyls (Aroclors® 1016 and 1254) on mortality,reproduction, and regeneration inHydra oligactis

Budding and tentacle regeneration were observed inHydra oligactis following exposure to the commercial PCB mixtures Aroclor 1016 and 1254. The acute toxicity of these PCB s was also compared by determining their respective 72-hr LC50s. The animals were exposed to the PCBs as a component of an artificial pond water medium with PCB dilutions being made from a 49:1, reagent grade acetone:PCB solution. All exposures were continuous with the medium being replaced daily. Animals used in studying the effects of these chemicals on tentacle regeneration had their heads transected at the time of initial exposure to the PCBs. Observation were taken at 24, 48, 72, and 96-hr of incubation at 19 ±0.2°C.Aroclor 1016 was more toxic than the more widely occurring 1254, with 72-hr LC50s of 5 mg/L and 20 mg/L, respectively. The two chemicals suppressed bud initiation equally, however, Aroclor 1254 had a significantly greater inhibitory effect on bud detachment when equal concentrations of the two PCBs were compared. As was the case with mortality, Aroclor 1016 had four times the suppressive effect of Aroclor 1254 on tentacle regeneration, with 50% inhibition occurring at 1 mg/L and 4 mg/ L, respectively. The quantitatively different effects on the two PCBs on mortality and tentacle regeneration may have been due to structural differences between the two PCBs or the different aqueous solubility properties (1016 being more soluble) of these chemicals. The results on bud initiation and detachment indicated that this developmental process may have been affected by the PCB through a different mechanism than was mortality and tentacle regeneration.Registered trademark, Monsanto Co., St. Louis, MO     

Induction of hepatic CYP1A activity as a biomarker for environmental exposure to Aroclor® 1254 in feral rodents

Specimens of the feral mouse species Reithrodontomys fulvescens trapped from a polychlorinated biphenyl (PCB)-contaminated field location had hepatic ethoxy-resorufin (ETR) O-dealkylase activities and immunoreactive CYP1A protein contents which were two- to threefold higher than those measured in animals of the same species and sex collected from non PCB-contaminated reference sites. Specimens with hepatic ETR O-dealkylase activities differing by as little as 50% could readily be assigned as originating from the PCB or reference sites by the use of a specific chemical inhibitor of cytochrome P450IA (CYP1A). The relative levels of ETR O-dealkylase activity in R. fulvescens significantly correlated with hepatic PCB burdens (r=0.819, P<0.01). When the magnitudes of the induced ETR O-dealkylase activities corresponding to given hepatic PCB burdens were compared between the feral animals, F344/NCr rats (Rattus norvegicus) or B6C3F1 mice (Mus musculus) exposed in the laboratory to dietary Aroclor^® 1254, the order of sensitivity to the inducing effects of PCBs were F344/NCr rat>B6C3F1 mouse>R. fulvescens.     

Immunological, Hematological, and Biochemical Responses in Immature White-Footed Mice Following Maternal Aroclor 1254 Exposure: A Possible Bioindicator

A number of hematological, immunological, and biochemical parameters were measured in Peromyscus leucopus pups born from dams exposed to a single dose (300 mg/kg body weight) of Aroclor 1254. To increase the chances of uncovering even modest consequences of the exposure, in one protocol the pups were weaned at 3 weeks and examined at 6 weeks of age, while in a second protocol the pups were kept with their mother for 4 weeks, at which time they were examined. The older pups showed significant decreases in body weight, ratio of spleen weight to body weight, numbers of peripheral white blood cells and lymphocytes, and number and percentage of monocytes. They also showed significant increases in the stimulation index in response to the mitogen phytohemagglutinin (PHA), percentage of peripheral blood neutrophils and liver EROD induction. Pups sacrificed at 4 weeks of age showed even more significant differences. Their body and liver weights, percentage and number of peripheral blood lymphocytes, and serum antibody titers were significantly lower than those of their controls, while spleen to body weight ratios, percent of neutrophils in their peripheral blood, and liver EROD, PROD, and BROD levels were significantly higher than those of the controls. The primary implication of this work is that white-footed mouse pups could be used as biomonitors of contaminated sites. Females could be captured at the sites and bred in captivity with normal males. The vulnerable parameters identified in this study could then be measured in the resulting offspring and compared with a database collected from normal pups. Received: 8 July 1998/Accepted: 13 December 1998     

Induction of hepatic cytochrome P4501A1/2B activity and disruption of thyroglobulin synthesis/secretion by mono-ortho polychlorinated biphenyl and its hydroxylated metabolites in rat cell lines

As the active metabolites of polychlorinated biphenyl (PCBs), hydroxylated polychlorinated biphenyls (OH-PCBs) are found in wildlife and human tissues. They have been proposed as main contributors for endocrine disruption of PCBs in living organisms. In this study, mono-ortho PCB 156 and its hydroxylated metabolites 4'-OH-PCB 159, 4'-OH-PCB 121, and 4'-OH-PCB 72 were selected to investigate the toxic effects on rat hepatoma H4IIE cell line and rat thyroid follicle FRTL-5 cell line at concentrations of 1, 10(2), 10(4) nM. 7-Ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) activities were determined with micro-EROD/PROD to indicate cytochrome P4501A1 (CYP1A1) and cytochrome P4502B (CYP2B) induction in the H4IIE cell after exposure for 72 h. To assess thyroid disruption of these compounds, thyroglobulin concentrations also were detected inside FRTL-5 cell with immunocellularchemistry and in its medium with radioimmunoassay after exposure for 24 h. Significant inductions of EROD activity by PCB156 at 10(2) and 10(4) nM (p < 0.05) were observed, but no effects by the three OH-PCBs in H4IIE cell line. 7-Pentoxyresorufin-O-dealkylase activities were induced only by 10(4) nM of PCB156 and the three OH-PCBs (p < 0.05). Meanwhile, significant increases of thyroglobulin concentrations were observed in the medium of FRTL-5 cell exposed to 4'-OH-PCB 121 and 4'-OH-PCB 72 at all of the test concentrations (p < 0.05), but not to the other compounds. The results demonstrated that mono-ortho PCBs mainly could be metabolized to hydroxylated metabolites through CYP1A1 instead of CYP2B. Moreover, after being metabolized, OH-PCBs still sustained the ability to induce PROD activity and did exhibit the disruption on thyroglobulin synthesis/excretion in rat cells.     

Exposure to the polychlorinated biphenyl mixture Aroclor 1254 alters melanocyte and tail muscle morphology in developing Xenopus laevis tadpoles

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that have damaging effects on both ecosystem and human health. Numerous studies have shown that exposure to PCBs can alter growth and development of aquatic organisms, including frogs. In this report, developing Xenopus laevis tadpoles were exposed to the PCB mixture Aroclor 1254. Tadpoles were exposed from 5 through 9 d postfertilization to either 0, 1, 10, 50, or 100 ppm Aroclor 1254. Exposure to an acute, high concentration of Aroclor 1254 (10, 50, and 100 ppm) caused statistically significant reductions in survival and body size. In addition, tadpoles exposed to these higher concentrations showed histological abnormalities, including aberrant tail tip, myotomal, and melanocyte morphologies. Described adverse health effects associated with PCB exposure of developing frogs will serve as useful health endpoints in ongoing and future molecular-based studies that correlate health effects with changes in gene expression.     

Potential ecotoxic effects of polychlorinated biphenyls on Xenopus laevis

We examined potential ecotoxic effects of polychlorinated biphenyl (PCB)3, PCB5, Aroclor 1254, and Aroclor 1242 on Xenopus laevis. Tadpoles were exposed to PCBs from stage 46/47 (system of Nieuwkoop and Faber) to the completion of metamorphosis. We demonstrated, to our knowledge for the first time, forelimb malformations caused by PCBs (malformation rate, > 70%). The malformed forelimbs were fixed in the adduction-backward rotation position and could not move. Therefore, malformed male frogs were destined to have no offspring, because they could not grasp the females with their forelimbs to mate. Alcian blue-alizarin red double-staining indicated that the forelimb malformation resulted from the shoulder abnormality. Compared with the normal shoulder joint, the proximal humerus with the humerus inter-rotated 90 degrees in the abnormal shoulder joint. Moreover, testes from more than a third of male frogs with exposed to PCBs exhibited feminization to different degrees at gross morphology and histology, with fewer or abnormal spermatogonia and oocytes. Gonadal abnormalities would lead directly to reproductive dysfunction and population decline. These results suggest that PCBs have potentially ecotoxic effects on amphibian populations. We infer that PCBs could play roles in amphibian malformations and population declines, at least at sites that are polluted heavily with PCBs.     

Assessment of the bioavailability of PAHs in rats exposed to a polluted soil by natural routes: induction of EROD activity and DNA adducts and PAH burden in both liver and lung.

In order to assess bioavailability of polycyclic aromatic hydrocarbons (PAHs) present in soils, male laboratory rats were exposed to litters of control and polluted soils. After 88+/-2 h of exposure, several biomarkers were measured in both liver and lung. When rats were exposed to SIV soil, contaminated by a mixture of at least 13 PAHs, (1) only 2 or 3 PAH compounds were detected in liver and lung; (2) cytochrome P450-dependent monooxygenase activity, followed by 7-ethoxyresorufin O-deethylase (EROD) activity measurement, was highly induced in liver (13-fold-induction) and lung (up to 78-fold); and (3) DNA adducts were significantly increased. For what concerns soil artificially contaminated by only one PAH (phenanthrene or B[a]P), EROD activity was not or fully induced, respectively. These results demonstrate the occurrence of a high bioavailability of PAHs to mammals in natural conditions of exposure. First results concerning DNA adducts must be profound, but they already show that a short exposure of mammals to PAH-polluted soils can lead to potential genotoxic effects. EROD activity can be used as a sensitive biomarker in both liver and lung of rats maintained on litters of soils in the laboratory, and such a test can be used routinely to contribute to risk assessment.     

Differential effects of two lots of aroclor 1254: congener-specific analysis and neurochemical end points.

Aroclor 1254 is a widely studied commercial polychlorinated biphenyl (PCB) mixture which, by definition, contains 54% chlorine by weight. Recent reports indicate substantial differences in the congener composition among Aroclor lots and hence their biologic effects. We designed the current study to compare the effects of two lots of Aroclor 1254 (lots 6024 and 124-191). We analyzed these two lots for PCB congeners, polychlorinated dibenzofurans (PCDFs), polychlorinated naphthalenes (PCNs), and polychlorinated dibenzodioxins (PCDDs). We used previously established techniques for analyzing intracellular Ca(2+) buffering and protein kinase C (PKC) translocation to test their biologic activity in neuronal preparations. PCB congener-specific analysis indicated that ortho and non-ortho congeners in these two lots varied in their percent contribution. Among all congeners, the percentages of non-ortho congeners (PCBs 77, 81, 126, and 169) were higher in lot 6024 (2.9% of total) than in lot 124-191 (0.02% of total). We detected no dioxins in these two lots (< 2 ppb). Although there are some differences in the congener composition, total PCNs were similar in both lots: 171 ppm in lot 6024 and 155 ppm in lot 124-191. However, total PCDFs were higher in lot 6024 (38.7 ppm) than in lot 124-191 (11.3 ppm). When we tested these two Aroclors on Ca(2+) buffering and PKC translocation in brain preparations, the effects were significantly different. Although lot 124-191 was more potent on PKC translocation than lot 6024, lot 6024 was slightly more active on Ca(2+) buffering than lot 124-191. These effects could not be attributed to the differences in the percentage of non-ortho congeners or PCDFs because they were inactive on these two parameters. The effects could not be attributed to PCNs because the levels were almost similar. The effects seen with two lots of Aroclor 1254 in neuronal cells were also not predicted based on the TCDD toxic equivalents (TEQs), although TEQs predicted the effects on ethoxyresorufin-O-deethylase (EROD) or methoxyresorufin-O-deethylase (MROD) activities. It is possible that the differential effects seen in neuronal cells could be caused by differences in the composition of ortho-congeners in these two mixtures, because PCBs with ortho-lateral substitutions can exhibit different activities on the selected neurochemical end points. Because of these differential effects with different lot numbers, the composition of Aroclor mixtures used in investigations should be disclosed.