Exposure to prenatal PCBs shifts the timing of neurogenesis in the hypothalamus of developing rats.

The developing brain is highly sensitive to the hormonal milieu, with gonadal steroid hormones involved in neurogenesis, neural survival, and brain organization. Limited available evidence suggests that endocrine-disrupting chemicals (EDCs) may perturb these developmental processes. In this study, we tested the hypothesis that prenatal exposure to a mixture of polychlorinated biphenyls (PCBs), Aroclor 1221, would disrupt the normal timing of neurogenesis in two hypothalamic regions: the ventromedial nucleus (VMN) and the preoptic area (POA). These regions were selected because of their important roles in the control of sociosexual behaviors that are perturbed in adulthood by prenatal EDC exposure. Pregnant Sprague-Dawley rats were exposed to PCBs from Embryonic Day 8 (E8) to E18, encompassing the period of neurogenesis of all hypothalamic neurons. To determine the birth dates of neurons, bromo-2-deoxy-5-uridine (BrdU) was administered to dams on E12, E14, or E16. On the day after birth, male and female pups were perfused, brains immunolabeled for BrdU, and numbers of cells counted. In the VMN, exposure to PCBs significantly advanced the timing of neurogenesis compared to vehicle-treated pups, without changing the total number of BrdU+ cells. In the POA, PCBs did not change the timing of neurogenesis nor the total number of cells born. This is the first study to show that PCBs can shift the timing of neurogenesis in the hypothalamus, specifically in the VMN but not the POA. This result has implications for functions controlled by the VMN, especially sociosexual behaviors, as well as for sexual selection more generally.

Endocrine-disrupting chemicals alter the neuromolecular phenotype in F2 generation adult male rats.

Endocrine-disrupting chemical (EDC) exposures to the fetus have long-lasting effects on health and disease in adulthood. Such EDC exposure to the F1 fetuses also reaches the germ cells that become the F2 generation. Previously, we demonstrated that adult social and communicative behaviors such as ultrasonic vocalizations and mating behaviors were altered by EDCs in F2 rats, especially males. In the current study, we used the brains of these F2 males to ascertain the underlying molecular changes in the hypothalamus related to these behavioral outcomes. Their progenitors were Sprague-Dawley rat dams, treated on pregnancy days 8 to 18 with one of three treatments: a polychlorinated biphenyl (PCB) mixture, Aroclor 1221, selected because it is weakly estrogenic; the anti-androgenic fungicide vinclozolin (VIN); or the vehicle, 6% dimethylsulfoxide in sesame oil (VEH). In adulthood, F1 male and female offspring were bred with untreated partners to generate paternal or maternal lineages of the F2 offspring, the subjects of molecular work. Quantitative real-time PCR was conducted in the medial preoptic area (POA) and the ventromedial nucleus (VMN) of the hypothalamus, selected for their roles in social and sexual behaviors. Of the genes assessed, steroid hormone receptors (estrogen receptor α, androgen receptor, progesterone receptor) but not dopamine receptors 1 and 2 or DNA methyltransferase 3a expression were altered, particularly in the VIN males. Several significant correlations between behavior and gene expression were also detected. These results suggest that preconceptional exposure of male rats to EDCs at the germ cell stage alters the neuromolecular phenotype in adulthood in a lineage-dependent manner.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Regulation of Liver Enriched Transcription Factors in Rat Hepatocytes Cultures on Collagen and EHS Sarcoma Matrices.

Liver-enriched transcription factors (LETF) play a crucial role in the control of liver-specific gene expression and for hepatocytes to retain their molecular and cellular functions complex interactions with extra cellular matrix (ECM) are required However, during cell isolation ECM interactions are disrupted and for hepatocytes to regain metabolic competency cells are cultured on ECM substrata. The regulation of LETFs in hepatocytes cultured on different ECM has not been studied in detail. We therefore compared two common sources of ECM and evaluated cellular morphology and hepatocyte differentiation by investigating DNA binding activity of LETFs at gene specific promoters and marker genes of hepatic metabolism. Furthermore, we studied testosterone metabolism and albumin synthesis to assess the metabolic competence of cell cultures. Despite significant difference in morphological appearance and except for HNF1ß (p<0.001) most LETFs and several of their target genes did not differ in transcript expression after Bonferroni adjustment when cultured on collagen or Matrigel. Nonetheless, Western blotting revealed HNF1ß, HNF3α, HNF3γ, HNF4α, HNF6 and the smaller subunits of C/EBPα and C/EBPß to be more abundant on Matrigel cultured cells. Likewise, DNA binding activity of HNF3α, HNF3ß, HNF4α, HNF6 and gene expression of hepatic lineage markers were increased on Matrigel cultured hepatocytes. To further investigate hepatic gene regulation, the effects of Aroclor 1254 treatment, e.g. a potent inducer of xenobiotic defense were studied in vivo and in vitro. The gene expression of C/EBP-α increased in rat liver and hepatocytes cultured on collagen and this treatment induced DNA binding activity of HNF4α, C/EBPα and C/EBPß and gene expression of CYP1A1 and CYP1A2 in vivo and in vitro. Taken collectively, two sources of ECM greatly affected hepatocyte morphology, activity of liver enriched transcription factors, hepatic gene expression and metabolic competency that should be considered when used in cell biology studies and drug toxicity testing.

Evaluation of estrogenic effects of polychlorinated biphenyls and organochlorinated pesticides using immature rat uterotrophic assay.

In this study, we investigated the effects of polychlorinated biphenyls (PCBs) and organochlorinated pesticides on the serum levels of luteinising hormone (LH), follicle stimulating hormone (FSH) and weights and histomorphometry of uterine tissue in immature female rats using uterotrophic assay. A total of 36 rats were randomly divided into six groups (n = 6 per group) as control, oestradiol (E2, 100 µg/kg), PCB 180, Aroclor 1221, endosulfan and mirex at 10 mg/kg dosage. After 3 days of injections (subcutaneous), animals were decapitated and blood samples were collected. Uteri were dissected, weighed out and then fixed in 10% formaldehyde. They were processed for histomorphometry. The serum levels of LH and FSH were determined by enzyme immunoassay. Uterine weight was significantly increased by E2 and reduced by mirex (p < 0.001 and p < 0.05, respectively). Total volume of uterus was significantly raised by E2, Aroclor 1221 and endosulfan compared with that of the control group (p < 0.01). The ratio of epithelium was significantly increased by E2, PCBs and pesticides (p < 0.01). The uterine cavity ratio was decreased by aroclor (p < 0.01), PCB 180 and mirex (p < 0.05). The serum levels of LH did not significantly differ among the groups but the levels of FSH were decreased by PCB 180 and endosulfan (p < 0.05 and p < 0.01, respectively). These findings suggest that PCB 180, Aroclor 1221 and endosulfan may be estrogenic in immature uterotrophic assay.

Impacts of molt-inhibiting organochlorine compounds on epidermal ecdysteroid signaling in the fiddler crab, Uca pugilator, in vitro.

Because of their chemical stability and lipophilicity, many organochlorine compounds (OCs) can readily accumulate in fatty tissues of crustaceans. Several OCs have been reported to inhibit crustacean molting. To determine whether the disruption of crustacean molting by these OCs involves interference with ecdysteroid signaling in the epidermis, the impacts of five molt-inhibiting OCs on the level of N-acetyl-beta-glucosaminidase (NAG, EC 3.2.1.30) mRNA in cultured epidermal tissues from the fiddler crab, Uca pugilator, were investigated using quantitative real-time PCR. The NAG mRNA was found to be inducible by 20-hydroxyecdysone (20-HE) in cultured epidermal tissues. The inducibility of NAG mRNA in cultured epidermal tissues by 20-HE is not only further direct evidence that epidermal expression of NAG gene in U. pugilator is controlled by the molting hormone but also validates the use of the NAG mRNA as a biomarker for epidermal ecdysteroid signaling. When Aroclor 1242, 2,4,5-trichlorobiphenyl (PCB29), endosulfan or kepone was administered alone, the expression of NAG gene in cultured epidermal tissues was upregulated, while heptachlor had no effects. Under binary exposure to both 20-HE and an OC, a condition similar to the natural hormonal milieu of epidermal tissues of animals impacted by OCs, both Aroclor 1242 and endosulfan were found to be capable of antagonizing ecdysteroid signaling in cultured epidermal tissues. This antagonizing effect on epidermal ecdysteroid signaling can at least partly explain the inhibitory effects of these two agents on crustacean molting. PCB29, when given together with 20-HE, produced an additive effect on epidermal ecdysteroid signaling but such an additive effect was not observed when kepone was combined with 20-HE.

In vitro and in vivo metabolism of the soy isoflavone glycitein.

Glycitein (GLY) is a major isoflavone of soy germ which is used as a functional ingredient to enrich foods with isoflavones as well as a component of soy supplements. Since data on the metabolism of GLY are incomplete, the in vitro phase I metabolism of GLY using rat liver microsomes, human liver microsomes (HLM), as well as human fecal flora was investigated. Furthermore, the in vivo metabolism has been studied after administration of GLY to Sprague-Dawley rats. The identification of the formed metabolites has been elucidated using HPLC/DAD, HPLC/API-ES MS, GC/MS, as well as reference compounds. With respect to the phase I metabolism, GLY has been converted to ten metabolites by liver microsomes of Aroclor-induced Wistar rats with 8-hydroxy(OH)-GLY as the main metabolite. HLM converted GLY to six metabolites with 8-OH-GLY and 6-OH-daidzein (DAI) being the major products. No sex-related differences were observed in each system. Intestinal metabolism of GLY led to four metabolites with 6-OH-DAI as the main product. The in vivo metabolism in rat urine after single-dose administration of GLY resulted in the identification of three oxidative and two bacterial metabolites as well as the demethylation product 6-OH-DAI as the main metabolite.

Effect of aroclor 1248 and two pure PCB congeners on phospholipase D activity in rat renal tubular cell cultures.

This paper elucidates the effect of different polychlorinated biphenyls (PCBs) on the phospholipase D (PLD) activity in soluble and particulate fractions of rat renal proximal tubular culture cells. Treatment with Aroclor 1248 (a commercial PCB mixture) caused a marked increase in the activity of PLD in intact renal tubular cells. The PLD activity was increased by Aroclor 1248 in the particulate fraction while the enzyme activity was unaffected in the soluble fraction. This work also shows that PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, a di-ortho-substituted nonplanar congener) can increase the activity of PLD only in the particulate fraction. The exposure of cell cultures to PCB 77 (3,3',4,4'-tetrachlorobiphenyl, a non-ortho-substituted planar congener) does not alter PLD activity. These results suggest that PCB effects are structure dependent. Therefore, in order to clarify the molecular mechanism of activation of PLD by PCBs, the contents of immunoreactive PLD were examined by immunoblot analysis. Renal tubular cells expressed a PLD protein of 120 kDa corresponding with the PLD1 mammalian isoform in both the particulate and the soluble fraction. Aroclor 1248, PCB 153, and PCB 77 do not induce changes in the levels of PLD protein. These data indicate that PCBs, particularly nonplanar congeners, increase PLD activity. Moreover, these changes could not be demonstrated in the enzyme content in rat renal tubular cell cultures.

Coping with polychlorinated biphenyl (PCB) toxicity: Physiological and genome-wide responses of Burkholderia xenovorans LB400 to PCB-mediated stress.

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.

Modulatory effects of Aroclors 1221 and 1254 on bone turnover and vertebral histology in intact and ovariectomized rats.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants. Two PCB mixtures, Aroclors 1221 and 1254 have been suggested to have estrogenic and anti-estrogenic properties, respectively. We have examined whether these PCB mixtures modulate bone turnover and vertebral histology in intact and ovariectomized (ovx) rat models. Thirty-two adult female rats were divided into four groups subcutaneously receiving 4% DMSO (control), A1221 (10 mg/kg), A1254 (10 mg/kg) oestradiol (E2, 30 microg/kg). These compounds were injected to the animals for a period of 6 weeks at two daily intervals. In the second model, rats (n=32) were ovx and allowed to recover for a period of 3 weeks. Control group received vehicle (4% DMSO) alone. Remaining rats were divided into three groups and injected (s.c.) with A1221, A1254 and E2 for 5 weeks. Urine samples were collected prior to end of the experiments. Then, all animals were decapitated. Serum parathyroid hormone (PTH), calcitonin and osteocalcin levels were determined by immunoradiometric method. Serum concentrations of alkaline phosphatase (ALP), calcium and inorganic phosphate were determined by enzymatic-colorimetric method. Urinary deoxypyridinoline (DPD) was measured by ELISA. Lumbar vertebrae (L2) of all animals were dissected out and processed for light microscopy. Levels of urinary DPD were significantly lowered in E2 -treated intact rats (p<0.001). Ovx significantly increased urinary DPD excretion (p<0.01) compared to intact control values. Administration of A1221 and A1254 had no significant effects in intact rats, however, they significantly reduced (p<0.05) and increased (p<0.001) urinary DPD levels in ovx rats, respectively. Neither of the PCB mixtures significantly changed serum osteocalcin and ALP levels in intact or ovx rats (except A1221 increased ALP in intact model, p<0.01). Both PCB mixtures had differential effects on serum PTH, calcitonin, calcium and inorganic phosphate concentrations. Treatment with A1221 reversed the adverse effects of ovariectomy on L2 histology. However, A1254 produced necrotic areas in vertebral bone, and this effect was expanded in ovx animals. Our findings suggest that both Aroclor compounds interfere with bone turnover mechanisms, particularly in ovx rats.

PCBs exert an estrogenic effect through repression of the Wnt7a signaling pathway in the female reproductive tract.

Polychlorinated biphenyls (PCBs) have been proposed to have a weak estrogenic activity and therefore pose a risk as potential environmental endocrine disruptors to the perinatal development of the female reproductive tract. Perinatal exposure to high concentrations of the potent synthetic estrogen diethylstilbestrol (DES) induces abnormal development of the female reproductive tract via a mechanism that acts through the down-regulation of Wnt7a (wingless-type MMTV integration site family, member 7A). To test the hypothesis that PCBs act as weak estrogens, we injected neonatal mice with a commercial PCB mixture (Aroclor 1254) or with low levels of DES and measured effects of exposure on Wnt7a expression and uterine morphology. We report here that neonatal PCB or low-level DES exposure resulted in the down-regulation of Wnt7a expression. In addition, both PCB and low-level DES exposure induced changes in the uterine myometrium and gland formation. These data reveal that weak estrogens such as the PCBs act through a Wnt7a-dependent pathway and suggest that Wnt7a regulation is a sensitive biomarker for testing weak estrogenic candidate compounds. The morphologic changes that were elicited by PCBs and DES were different immediately after exposure, suggesting that Wnt7a-independent pathways are also activated by one or both of these compounds. Although Wnt7a down-regulation is transient after estrogenic exposure, subsequent morphologic changes became more pronounced during postnatal and adult life, suggesting that the female reproductive tract is permanently reprogrammed after exposure even to weak estrogenic compounds. In addition, Wnt7a heterozygous mice were more sensitive to PCB exposure, revealing an important genetic predisposition to risks of environmental endocrine disruptors.

Structural elucidation of hydroxylated metabolites of the isoflavan equol by gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry.

Equol has, as have other isoflavonoids, recently gained considerable interest due to its possible health effects. However, detailed studies on the metabolism of equol are scarce. Therefore, we investigated the phase I metabolism of equol using liver microsomes from Aroclor-treated male Wistar rats as well as from a male human. The identification of the metabolites formed was elucidated using high performance liquid chromatography (HPLC) with diode array detection, HPLC/atmospheric pressure ionization electrospray mass spectrometry, and gas chromatography-mass spectrometry, as well as reference compounds. (+/-)-Equol was converted to 11 metabolites by the liver microsomes from Aroclor-pretreated rats comprising three aromatic monohydroxylated and four aliphatic monohydroxylated as well as four dihydroxylated products. The main metabolite was identified as 3'-hydroxy-equol. Using human liver microsomes, equol was converted to six metabolites with 3'-hydroxy- and 6-hydroxy-equol as main products. Furthermore, the aliphatic hydroxylated metabolite 4-hydroxyequol, which was recently detected in human urine after soy consumption, was formed. On the basis of these findings, it is suggested that phase I metabolism of equol is part of a complex biotransformation of the soy isoflavone daidzein in humans in vivo.

Genotoxicity of the Musi River (Hyderabad, India) investigated with the VITOTOX test.

The bacterial VITOTOX genotoxicity test was used to screen water samples collected from three different stations along the banks of the river Musi, in Hyderabad, India. Water was collected at three stations that differed from each other in the nature of the surrounding industrial and other activities. A number of different pollutants were also measured in water, soil and air samples. The three stations were found highly polluted and different with regard to the genotoxicity and toxicity of their samples. These results demonstrate the need for further biological studies in this area to generate valuable data on genomic instability, risk assessment of cancer, and to provide avenues for risk management.

Endocrine disruptive effects of polychlorinated biphenyls on the thyroid gland in female rats.

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants. Aroclor 1221 (A1221) and Aroclor 1254 (A1254) are commercial PCB mixtures with low and high number of chlorination, respectively. We have comparatively investigated effects of A1221 and A1254 on serum levels of thyroid hormones and thyroid gland histology in adult female Wistar rats. Animals were subcutaneously injected with A1221 (10 mg/kg) or A1254 (10 mg/kg) for six weeks. One group of animals served as control. At the end, all animals were decapitated and trunk blood collected. Serum levels of triiodothyronine (T3) and thyroxine (T4) were measured by the electrochemiluminescence immunoassay method. Thyroid glands were removed for histopathological examination under light microscopy. Serum total T4 levels were significantly increased in A1221- and A1254-treated rats (p < 0.05). Serum free T4 levels were significantly increased in the A1254-treated rats (p < 0.01), but not in the A1221-treated rats. In contrast, the treatment with A1221 caused a significant increase in serum free T3 concentrations (p < 0.05) but not with A1254. Notably, either A1221 or A1254 caused distinct histopathological changes, such as formation of many microfollicles in the thyroid gland, which mimic the changes seen in thyrotoxicosis. In conclusion, both PCB mixtures induce toxic effects in the thyroid gland regardless of their degree of chlorination. We suggest that these environmental contaminants may disrupt thyroid hormone homeostasis in exposed individuals and thus pose a threat to human health.

The inhibition of LPS-induced splenocyte proliferation by ortho-substituted and microbially dechlorinated polychlorinated biphenyls is associated with a decreased expression of cyclin D2.

Immunological effects of polychlorinated biphenyls (PCBs) have been demonstrated in our laboratories with the peferential inhibition of lipopolysaccharide (LPS)-induced splenocyte proliferation by ortho-substituted PCB congeners. An investigation of the mechanism behind this immunotoxicity revealed an interruption in the progression of murine lymphocytes from G0/G1 into S phase by Aroclor 1242 and the di-ortho-substituted congener, 2,2'-chlorobiphenyl (CB), whereas, a non-ortho-substituted congener, 4,4'-CB, did not affect cell cycle progression. This interruption of cell cycle progression by 2,2'-CB and Aroclor 1242 was associated with a decreased expression of the cell cycle regulatory protein, cyclin D2, while expression was not affected by exposure to the non-ortho-substituted 4,4'-CB. These results suggest the preferential inhibition of LPS-induced splenocyte proliferation by ortho-substituted congeners is a result of a decreased expression of cyclin D2, which leads to an interruption in cell cycle progression. In addition, PCB mixtures with an increased percentage of chlorines in the ortho position following an environmentally occurring degradation process inhibited LPS-induced proliferation, interrupted cell cycle progression, and decreased cyclin D2 expression. This study provides evidence for a mechanism of action of the immunological effects of ortho-substituted individual congeners as well as environmentally relevant mixtures enriched in congeners with this substitution pattern.

Use of cultured precision-cut rat lung slices to study the in vitro induction of pulmonary cytochrome P450 forms.

1. The aim was to investigate the effects of some cytochrome P450 (CYP) enzyme inducers on CYP1A and CYP2B subfamily forms in cultured precision-cut rat lung slices. 2. Precision-cut lung slices were prepared from male Sprague-Dawley rats and cultured for 24 and/or 48 h in medium containing 0-20 micro g ml(-1) Aroclor 1254 (ARO), 0-50 micro M beta-naphthoflavone (BNF) and 0-50 micro M benzo(a)pyrene (BP). 3. Treatment with ARO, BNF and BP produced significant increases in lung slice whole homogenate 7-ethoxyresorufin O-deethylase activity. 4. Levels of CYP1A1 apoprotein were markedly increased in lung slice microsomes after treatment for 48 h with either 10 micro g ml(-1) ARO or 5 micro M BNF. In contrast, neither ARO nor BNF had any marked effect on levels of CYP2B1/2 apoprotein in 48-h cultured rat lung slice microsomes. 5. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan) was used to quantify lung slice CYP1A1 and CYP2B1/2 mRNA levels. Rat lung slice CYP1A1 mRNA levels were increased up to 8.3-fold after treatment for 24 h with 2 and 10 micro g ml(-1) ARO, 0.5 and 5 micro M BNF, and 20 micro M BP. In contrast, treatment with 10 micro g ml(-1) ARO produced only a small 1.6-fold increase in CYP2B1/2 mRNA levels. 6. Precision-cut lung slices are a useful model in vitro system for the assessment of the effects of chemicals on pulmonary CYP forms.

Differential expression of CYP1A1 mRNA in gill, intestine and liver of tilapia fed with PCB Aroclor-1254 and Aroclor-1260 spiked food.

The differential expression of cytochrome P4501A1 gene expression (CYP1A1) in liver, intestine and gill of juvenile tilapia following oral exposure to polychlorinated biphenyl (PCB) Aroclor-1254 or Aroclor-1260 spiked food were investigated. The fish was fed with 0.1 g/g of body weight each day of the spiked food with a concentration of approximately 8 ppm. Fish fed with betaNF- or acetone-spiked food were used as positive and negative controls, respectively. On day 1, 3 and 7 of post-treatment, the fish were sampled and analyzed for the total accumulated levels of PCBs, as well as the induced levels of CYP1A1 mRNA. Our results indicated that the fish fed with PCBs-spiked food showed a time-dependent bioaccumulation of PCBs. The time course studies also revealed that in the first 3 days of feeding, significant increases in CYP1A1 were found in intestine (betaNF: 320% and Aroclor-1260: 290%), followed by liver (betaNF: 144%, Aroclor-1260: 123% and Aroclor-1254: 110%). The transcript levels, then declined slowly. There was no induction of gill CYP1A1 mRNA expression in all treatment groups. Among all the tested tissues, the highest intestinal CYP1A1 transcripts induction has highlighted its prompt response to dietary PCBs exposure. Our study supported the important role of intestine in response to dietary PCBs exposure. In addition, in the present study the relative potency of Aroclor-1260 over Aroclor-1254 in stimulating CYP1A1 expression was discussed, indicating that the highly chlorinated PCBs had a higher potency of bioaccumulation and CYP1A1 mRNA induction.

Use of a quantitative real-time reverse transcription-polymerase chain reaction method to study the induction of CYP1A, CYP2B and CYP4A forms in precision-cut rat liver slices.

1. The aim was to employ real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) technology (TaqMan to examine the induction of some selected cytochrome P450 (CYP) forms in precision-cut rat liver slices. 2. Taqman primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1 and CYP4A1 forms. 3. Rat liver slices were cultured in control medium or medium containing either 10 micro g ml(-1) Aroclor 1254 (ARO), 500 micro M sodium phenobarbitone (NaPB) or 50 micro M Wy-14643 (WY) for 3, 6 and 24 h. 4. Compared with control liver slices, treatment with ARO for 3 and 6 h produced 24- and 184-fold increases, respectively, in CYP1A1 mRNA levels, and after 24h produced an 85-fold increase in CYP1A2 mRNA levels. Levels of CYP1A1 and CYP1A2 mRNA were not markedly affected by NaPB and WY. 5. Treatment with ARO and PB for 24 h produced 10.6- and 23.8-fold increases, respectively, in CYP2B1 mRNA. Levels of CYP2B1 mRNA were not markedly affected by WY. 6. Treatment with WY, but not ARO and NaPB, for 24h produced a 20.4-fold increase in levels of CYP4A1 mRNA. 7. These results demonstrate that cultured liver slices may be used to evaluate the effect of xenobiotics on CYP form mRNA levels.

Effects of furazolidone, PCB77, PCB126, Aroclor 1248, paraquat and p,p'-DDE on transketolase activity in embryonal chicken brain.

The effect of in ovo exposure to PCBs, DDE and paraquat on transketolase activity was measured in 19-day-old chicken embryos. Furazolidone was used as a positive control for decreased activity of the enzyme. The potency of contaminants to interact with transketolase was also tested in an in vitro system, using control brain 7000xg supernatants containing the enzyme. No effects were found on transketolase activity after in ovo or in vitro exposure to PCB126, Aroclor, DDE or paraquat. PCB77 decreased transketolase activity in vitro, but only at concentrations that, extrapolated to in ovo exposure, would be lethal to the embryo. Furazolidone decreased transketolase activity both in ovo and in vitro. For this contaminant, thiamine residues were analysed in the yolk sacs, but no differences were found between exposed and non-exposed eggs. Transketolase is dependent on thiamine pyrophosphate as a cofactor, and therefore, the decreased enzyme activity could be the result of an interaction between furazolidone and thiamine metabolism. Since thiamine residues were not affected by furazolidone and transketolase inhibition in vitro was similar to the inhibition after in ovo exposure, it was concluded that furazolidone interacted with transketolase on the enzymatic level rather than by a depletion of thiamine.

In vitro testing for genotoxicity of the herbicide terbutryn: cytogenetic and primary DNA damage.

Terbutryn is a widely used preemergence and postemergence s-triazine herbicide. This pesticide is used in agriculture as a control agent for most grasses and many annual broadleaf weeds in cereal and legume fields, and under fruit trees. Unexpectedly, this compound was found to persist in the environment (240 and 180 days in pond and river sediment, respectively) and to have the tendency to move from treated soils to water compartments through water runoff and leaching. However, only scant information is available about the genotoxic properties of terbutryn. In the present in vitro study, we investigated the relationship between cytogenetic damage, as evaluated in the sister-chromatid exchange (SCE) assay and the micronucleus (MN) test, and primary DNA damage (as evaluated by the "comet" assay). Cytogenetic and primary DNA damage were recorded in vitro in freshly isolated human peripheral blood leukocytes. Our results showed that the tested compound failed to produce any significant increases in SCE or MN, neither in the absence nor in the presence of S9-mix. However, terbutryn was found to induce primary DNA damage, more pronounced without S9 mix, even though in the absence of a clear trend for dose-dependence and in the presence of a concomitant mild cytotoxic effect.