RESUMEN
Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is implicated in many developmental and behavioral adverse outcomes in offspring of exposed parents. The objective of this study was to investigate sex-dependent multigenerational effects of preconceptional effects of BaP exposure. Adult wild-type (5D) zebrafish were fed 708 µg BaP/g diet (measured) at a rate of 1% body weight twice/day (14 µg BaP/g fish/day) for 21 days. Fish were spawned using a crossover design, and parental (F0) behavior and reproductive indexes were measured. In offspring, behavioral effects were measured at 96 h post fertilization (hpf) in F1 & F2 larvae, and again when F1s were adults. Compared to controls, there was no significant effect on F0 adult behavior immediately following exposure, but locomotor activity was significantly increased in F1 adults of both sexes. Larval behavior (96 hpf, photomotor response assay) was significantly altered in both the F1 and F2 generations. To assess molecular changes associated with BaP exposure, we conducted transcriptome and DNA methylation profiling in F0 gametes (sperm and eggs) and F1 embryos (10 hpf) from all four crosses. Embryos resulting from the BaP male and control female cross had the most differentially expressed genes (DEGs) and differentially methylated regions (DMRs). Some DMRs were associated with genes encoding chromatin modifying enzymes suggesting regulation of chromatin conformation by DNA methylation. Overall, these results suggest that parental dietary BaP exposure significantly contributes to the multigenerational adverse outcomes.
Asunto(s)
Metilación de ADN , Exposición Paterna , Animales , Femenino , Masculino , Benzo(a)pireno/toxicidad , Benzo(a)pireno/metabolismo , Estudios Cruzados , Expresión Génica , Exposición Paterna/efectos adversos , Semen , Pez Cebra/metabolismoRESUMEN
The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.
Asunto(s)
Dependovirus/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Gatos , Dependovirus/genética , Femenino , Terapia Genética , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes/genética , Inyecciones Intraoculares , Masculino , Ratones , Células Fotorreceptoras Retinianas Conos/virología , Células Fotorreceptoras Retinianas Bastones/virología , Transducción Genética , Tropismo ViralRESUMEN
Benzo[a]pyrene (BaP) is an established carcinogen and reproductive and developmental toxicant. BaP exposure in humans and animals has been linked to infertility and multigenerational health consequences. DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and mapping of methylation patterns has become an important tool for understanding pathologic gene expression events. The goal of this study was to investigate aberrant changes in promoter DNA methylation in zebrafish embryos and larvae following a parental and continued embryonic waterborne BaP exposure. A total of 21 genes known for their role in human diseases were selected to measure percent methylation by multiplex deep sequencing. At 96hpf (hours post fertilization) compared to 3.3hpf, dazl, nqo1, sox3, cyp1b1, and gstp1 had higher methylation percentages while c-fos and cdkn1a had decreased CG methylation. BaP exposure significantly reduced egg production and offspring survival. Moreover, BaP decreased global methylation and altered CG, CHH, and CHG methylation both at 3.3 and 96hpf. CG methylation changed by 10% or more due to BaP in six genes (c-fos, cdkn1a, dazl, nqo1, nrf2, and sox3) at 3.3hpf and in ten genes (c-fos, cyp1b1, dazl, gstp1, mlh1, nqo1, pten, p53, sox2, and sox3) at 96hpf. BaP also induced gene expression of cyp1b1 and gstp1 at 96hpf which were found to be hypermethylated. Further studies are needed to link aberrant CG, CHH, and CHG methylation to heritable epigenetic consequences associated with disease in later life.
Asunto(s)
Benzo(a)pireno/toxicidad , Metilación de ADN , Embrión no Mamífero/efectos de los fármacos , Regiones Promotoras Genéticas , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/embriología , Animales , Embrión no Mamífero/metabolismo , Femenino , Larva/efectos de los fármacos , Larva/metabolismo , Masculino , Pez Cebra/genéticaRESUMEN
Rodent models of retinal angiogenesis play a pivotal role in angiogenesis research. These models are a window to developmental angiogenesis, to pathological retinopathy, and are also in vivo tools for anti-angiogenic drug screening in cancer and ophthalmic research. The mouse model of oxygen-induced retinopathy (OIR) has emerged as one of the leading in vivo models for these purposes. Many of the animal studies that laid the foundation for the recent breakthrough of anti-angiogenic treatments into clinical practice were performed in the OIR model. However, readouts from the OIR model have been time-consuming and can vary depending on user experience. Here, we present a computer-aided quantification method that is characterized by (i) significantly improved efficiency, (ii) high correlation with the established hand-measurement protocols, and (iii) high intra- and inter-individual reproducibility of results. This method greatly facilitates quantification of retinal angiogenesis while at the same time increasing lab-to-lab reproducibility of one of the most widely used in vivo models in angiogenesis research.
Asunto(s)
Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Neovascularización Retiniana/diagnóstico por imagen , Algoritmos , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Eficiencia , Fluorescencia , Ratones , Neovascularización Patológica/diagnóstico por imagen , Variaciones Dependientes del Observador , Oxígeno , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/patologíaRESUMEN
This research addresses whether chemical contaminants are having a biologically measurable effect on largemouth bass (Micropterus salmoides), an important fish species in the Mobile Bay National Estuary. Bass and sediment samples were collected in September 2001 from Big Bateau and D'Olive Bays; the latter has been suggested to be an environmentally impacted site. There was a significant difference in the age distribution of bass collected from the two sites. However, none of the animals showed any evidence of reproductive activity or had mature gametes. The age and gonadosomatic index were greater in animals collected from the Bateau site. Fish liver microsome ethoxyresorufin-O-deethylase (EROD) activities (elevated in the presence of CYP1A-inducing contaminants) were not significantly different between sites but were significantly higher in older bass. Plasma steroid concentrations in younger animals (less than 1 year old) tended to be higher and more variable than the concentrations found in older animals. Once the significant effects of age on plasma steroid concentrations were removed, an analysis of the residual change in steroids revealed no differences in testosterone or estradiol between animals collected at the two sites. Sediments were collected for chemical extraction and analysis in two bioassays: the H4IIE rat hepatoma EROD assay and the yeast estrogen screen (YES assay). The H4IIE bioassay indicated the presence of sediment BaP induction equivalents between 72 and 320 ng/g, whereas the YES assay indicated that potent estrogenic substances were not present in the sediment samples. The H4IIE bioassay results were higher for D'Olive samples, which was consistent with higher PAH concentrations in those sediments. Taken together the biomarkers and chemical analysis suggested generally low organic contamination at these two sites.
Asunto(s)
Lubina/crecimiento & desarrollo , Monitoreo del Ambiente , Sedimentos Geológicos/química , Contaminantes Químicos del Agua/análisis , Alabama , Animales , Lubina/metabolismo , Unión Competitiva , Bioensayo , Citocromo P-450 CYP1A1/metabolismo , Estradiol/sangre , Humanos , Metales Pesados/análisis , Metales Pesados/toxicidad , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Hidrocarburos Policíclicos Aromáticos/análisis , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Estrógenos/metabolismo , Saccharomyces cerevisiae/genética , Testosterona/sangre , Contaminantes Químicos del Agua/toxicidadRESUMEN
Certainpolynuclear aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) induce CYP1A-dependent enzyme activities. Because PAHs are ubiquitous environmental contaminants, and some are aryl hydrocarbon agonists, CYP1A has been used as a biomarker for PAH exposure. However, PAHs exist in the environment in complex mixtures that may confound biomarker results. In in vitro studies, the PAH fluoranthene (FL) failed to increase or enhance CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity in cells, but rather inhibited activities induced by AhR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(k)fluoranthene. In order to determine the in vivo effects of FL on CYP1A and DNA adduct levels, Fundulus heteroclitus were given single ip injections of either BaP (5 mg/kg), BaP + FL (5 mg/kg each), BaP + FL (5 and 50 mg/kg, respectively), FL (5 mg/kg), FL (50 mg/kg), or corn oil control. BaP-treated fish had liver microsome EROD activities significantly higher than controls, whereas FL-treated fish were not different from controls. EROD activities in BaP + FL cotreatments were significantly lower compared to fish treated with BaP alone. When FL was incubated with BaP-induced microsomes, the IC50 for inhibition of EROD activity was 1.4 x 10(-5) M FL. Kinetic studies indicated a significant noncompetitive component to the FL inhibition. When fish were treated with [(14)C]FL, the concentration of radiolabel associated with microsomal preparations was four orders of magnitude lower than the IC50. Therefore, the presence of FL or a FL metabolite was insufficient to account for the inhibition by a kinetic mechanism. In contrast to cell studies, CYP1A immunoreactive protein was significantly decreased in vivo by FL cotreatment, indicating that FL may inhibit EROD activity by down-regulating the CYP1A protein. A covalent interaction of [(14)C]FL with CYP1A was not detected. Total (32)P-postlabeled DNA adducts were not significantly changed by cotreatment of FL and BaP; however, cotreatment with 50 mg/kg FL decreased one adduct and increased another significantly. Because FL and perhaps other inhibitory PAHs, co-occur in the environment with CYP1A inducers, CYP1A-dependent bioassays may cause an underestimation of PAH exposures.
Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Inhibidores Enzimáticos/farmacología , Fluorenos/farmacología , Fundulidae/metabolismo , Hidrocarburos Policíclicos Aromáticos/farmacología , Animales , Benzo(a)pireno/farmacología , Unión Competitiva/efectos de los fármacos , Aductos de ADN/análisis , Aductos de ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Hígado/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Dibenzodioxinas Policloradas/farmacologíaRESUMEN
Since cyclodextrin gas chromatography columns became popular for chiral separations, many researchers have noticed high enantiomeric ratios [ER: (+)-enantiomer/(-)-enantiomer] for alpha-HCH in the brains of wildlife. This investigation used the laboratory rat as a model for these phenomena. Rats were either pretreated with phenobarbital (PB) or left untreated and then dosed with alpha-HCH. Animals were sacrificed after 1 or 24 h. The ER averaged 0.95 +/- 0.01 in blood, 1.29 +/- 0.02 in fat, and 0.77 +/- 0.004 in liver. ERs in brain ranged from 2.8 +/- 0.5 to 13.5 +/- 0.4. Both the tissue concentration distribution and the ERs agree well with those previously reported in wildlife. To determine whether high brain ERs were due to enantioselective metabolism or transport through the blood-brain barrier, alpha-HCH exposed brain and liver tissue slices were compared. Concentrations in the brain slices did not decrease with PB pretreatment but did decrease in the liver slices. Enantiomeric ratios in the brain slices averaged 1.11 +/- 0.02 and were 0.76 +/- 0.03 in liver slices for the PB pretreated rats. These data indicate that the enantioselective metabolism of alpha-HCH by the brain is not the mechanism responsible for high ERs in this tissue.
Asunto(s)
Encéfalo/metabolismo , Hexaclorociclohexano/química , Hexaclorociclohexano/farmacocinética , Hígado/metabolismo , Tejido Adiposo/química , Tejido Adiposo/metabolismo , Animales , Química Encefálica , Contaminantes Ambientales/análisis , Hexaclorociclohexano/análisis , Humanos , Técnicas In Vitro , Hígado/química , Masculino , Modelos Animales , Fenobarbital/farmacología , Ratas , Ratas Sprague-Dawley , Phocidae , Estereoisomerismo , BallenasRESUMEN
DNA repair is a critical process in protecting cellular genetic information from mutation. Nucleotide excision repair (NER) is a mechanism by which cells correct DNA damage caused by agents that form bulky covalent adducts and UV photoproducts such as thymine dimers and 6-4 photoproduct. NER, sometimes called dark repair, is generally accepted as being low in fish compared to mammals. This study was designed to quantitate NER in two related catfish species that have known differential sensitivities to liver carcinomas. The original hypothesis was that the more cancer resistant species, channel catfish (Ictalurus punctatus), would have more efficient DNA repair compared to the more sensitive brown bullhead (Ameriurus nebulosus). In order to measure NER, primary cultured hepatocytes of both species were exposed to UV light (10-40 J/m2) and collected at 0, 24, 48 and 72 h after exposure. Total DNA was extracted from the cells and incubated with T4 endonuclease V. Using alkaline gel electrophoresis, endonuclease sensitive sites (ESS) were quantified. Results from the ESS assay indicated there was a UV dose-response increase in thymine dimers from 0 to 40 J/m2. However, no repair (decrease in number of ESS) occurred in either fish species over a 72-h time period. When cells were exposed to photoreactivating fluorescent light, repair was detected. These studies highlight the difficulty of measuring NER in fish and are consistent with the low levels of NER reported by other researchers in fish.
Asunto(s)
Reparación del ADN , Hepatocitos/efectos de la radiación , Ictaluridae/genética , Rayos Ultravioleta/efectos adversos , Proteínas Virales , Animales , Técnicas de Cultivo de Célula , Desoxirribodipirimidina Fotoliasa/farmacología , Desoxirribonucleasa (Dímero de Pirimidina) , Relación Dosis-Respuesta en la Radiación , Endodesoxirribonucleasas/farmacología , Masculino , Dímeros de Pirimidina/efectos de la radiación , Factores de TiempoRESUMEN
Differential susceptibility of polycyclic aromatic hydrocarbon (PAH)-mediated liver cancer exists in two related species of Ictalurid catfish. Two hypotheses are addressed in this study to explain this difference. Specifically, the relatively insensitive channel catfish 1) do not produce mutagenic PAH metabolites, and/or 2) they more quickly eliminate PAHs due to greater Phase II enzyme activities than the more sensitive brown bullhead. Livers and bile were collected from each species 6, 24, 72, and 168 h after a single 10 mg/kg i.p. benzo(a)pyrene (BaP) exposure. BaP treatment had no significant effect on cytosolic 1-chloro-2,4-dinitrobenzene or ethacrynic acid (EA)-glutathione-S:- transferase (GST) and cis-stilbene oxide-microsomal epoxide hydrolase (EH) activities of either species. Channel catfish EH and GST activities were 1.2-fold higher than brown bullhead activities (p = 0.058 and p < 0.002, respectively). HPLC-APCI-MS of extracted bile and bile enzymatically digested to detect glucuronyl transferase (GT), GST, and sulfotransferase (ST) conjugated metabolites indicated no species differences in elimination or profiles of total biliary metabolites. GT conjugates predominated; ST and GST conjugates were minimal. BaP-diones accounted for the majority of metabolites in both species. Overall, these results indicated that brown bullhead preferentially formed BaP-7,8-dihydrodiol, a precursor to the DNA-reactive BaP-7, 8-dihydrodiol-9,10-epoxide (BPDE), which may be linked to the increased PAH susceptibility in this species.
Asunto(s)
Benzo(a)pireno/metabolismo , Carcinógenos/metabolismo , Ictaluridae/metabolismo , Animales , Benzo(a)pireno/toxicidad , Bilis/metabolismo , Carcinógenos/toxicidad , Citocromo P-450 CYP1A1/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , ADN/efectos de los fármacos , Aductos de ADN/efectos de los fármacos , Dinitroclorobenceno/metabolismo , Epóxido Hidrolasas/metabolismo , Ácido Etacrínico/metabolismo , Glutatión Transferasa/metabolismo , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Especificidad de la EspecieRESUMEN
Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.
Asunto(s)
Citocromo P-450 CYP1A1/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fluorenos/farmacología , Animales , Bioensayo , Citocromo P-450 CYP1A1/biosíntesis , Aductos de ADN/aislamiento & purificación , ADN de Neoplasias/aislamiento & purificación , Inducción Enzimática , Femenino , Modelos Lineales , Microsomas Hepáticos/enzimología , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
The H4IIE rat hepatoma cell bioassay has been extensively used to assess the toxic equivalents (TEQs) of complex mixtures of halogenated aromatic hydrocarbons in environmental samples. However, there is often a discrepancy between bioassay induction results and toxic equivalents calculated from chemical analysis of samples; the former generally yield higher bioassay-TEQs. Polynuclear aromatic hydrocarbons (PAHs) are a class of chemicals which can significantly contribute to induction-TEQs. Benzo(a)pyrene (BAP), dibenz(a, h)anthracene (DBA), benz(a)anthracene (BA), benzo(k)fluoranthene (BkF), benzo(b)fluoranthene (BbF), chrysene (Chr), and indeno(1,2,3-c,d) pyrene (IdP) are carcinogenic PAHs found in environmental samples, including oysters collected from Galveston Bay. The induction potency of these PAHs relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was determined individually in rat hepatoma H4IIE cells seeded in 6-well plates, and the induction-derived equivalency factors (EFs) relative to TCDD were 0. 000354, 0.00203, 0.000025, 0.00478, 0.00253, 0.00020, 0.0011 for BAP, DBA, BA, BkF, BbF, Chr, and IdP, respectively. Dilutions of a reconstituted PAH mixture containing 23 PAHs (744 to 4466 ng/g total PAHs) with constant percentages of BAP (4.5%), DBA (3.5%), BA (2.4%), BkF (3.7%), BbF (3.5%), Chr (4.7%), and IdP (4.2%) yielded bioassay-derived induction-EQs that ranged from 0.52 to 1.44 ng/g. Oysters exposed in the laboratory to the same PAH mixture for 30 days differentially accumulated the PAHs with time. Bioassay-EQs for these oyster extracts ranged from 0.94 to 5.79 ng/g. These results were similar to the chemically calculated EQs which varied from 0.81 to 3.13 ng/g.
Asunto(s)
Carcinógenos/toxicidad , Contaminantes Ambientales/toxicidad , Hígado/efectos de los fármacos , Hidrocarburos Policíclicos Aromáticos/toxicidad , Animales , Bioensayo , Carcinógenos/química , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Citocromo P-450 CYP1A1/metabolismo , Inducción Enzimática , Hígado/citología , Hígado/enzimología , Ostreidae/efectos de los fármacos , Ostreidae/metabolismo , Hidrocarburos Policíclicos Aromáticos/química , RatasRESUMEN
Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P4501B1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 microM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1A1 mRNA levels, a response abolished by cotreatment with 10 microM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with alpha-naphthoflavone (alpha NF, 1 microM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 microM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 microM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with alpha NF (1 microM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Riñón/metabolismo , ARN Mensajero/metabolismo , Animales , ADN/biosíntesis , Activación Enzimática , Femenino , Ratas , Ratas Sprague-DawleyRESUMEN
Halogenated hydrocarbons such as polychlorinated biphenyls (PCB's), heptachlor (HEP), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane (DDT), and pentachlorophenol (PCP) are environmental contaminants and, at times, can bioaccumulate in the food chain. Cattle have been contaminated in a variety of ways, but generally it is believed that they are only affected by high concentrations of the chemicals. Rumen microorganisms, however, may be affected at lower doses, thus possibly affecting the cow's growth and milk production. Polychlorinated biphenyls, HEP, DDT, and PCP were tested by a 1-stage in vitro fermentation procedure. Substrate utilization was determined by measuring percent dry matter disappearance. Four concentrations (0, 10, 50, and 100 ppm) were studied, and in vitro incubations were conducted for 24 and 48 hr. Samples were removed from 48-hr incubations to determine if the chlorinated hydrocarbons were metabolized during fermentation. Dry matter disappearance proved to be a reliable method to determine microbial activity in the presence of chemicals. Substrate dry matter disappearance for controls and all concentrations of PCB's, HEP, and DDT was approximately 50 and 80% at 24 and 48 hr, respectively. The PCP significantly (P less than 0.05) depressed the percent dry matter disappearance in 50- and 100-ppm cultures to 45 and 30% at 24 hr and 70 and 50% at 48 hr, respectively. Metabolic changes in the test chemicals were not detected by gas chromatographic analysis.