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Male mice were treated with structurally diverse herbicides to study their effect on liver xenobiotic-metabolizing enzymes. Chlorfiurecol, trifluralin, alachlor, propham, MCPP and 2,4-DP caused increases in phase I (cytochrome P-450, ethoxycoumarin O-deethylase, and/or aminopyrine N-demethylase) and phase II (microsomal epoxide hydrolase and cytosolic glutathione S-transferase) activities. MCPP and 2,4-DP also increased cytosolic epoxide hydrolase and carnitine acetyltransferase activities suggestive of peroxisome proliferation. Benthiocarb and molinate increased only some phase II enzyme activities. Dicamba, at the dose employed, caused mortality and decreases in some of the enzymes monitored. Most of the herbicides tested induced xenobiotic-metabolizing enzyme activities, the pattern of induction being dependent on herbicide structure.

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The capacity of the testis to metabolize xenobiotics has been proposed to play a role in the susceptibility of different species to testicular toxicity. Since species differences in testicular xenobiotic metabolizing enzyme activities are not well documented, the primary objective of the present study was to compare enzyme activities in subcellular fractions prepared from rat, mouse, monkey, and human testes. In microsomal fractions, enzyme activities measured were pentoxyresorufin O-dealkylase (PROD), ethoxyresorufin O-dealkylase (EROD), and epoxide hydrolase (mEH). In cytosolic preparations, epoxide hydrolase (cEH) and glutathione S-transferase (cGST) activities were measured. PROD activity was not detectable in any of the species studied, while it was readily detected in liver microsomes used as a positive control. Although EROD activity was low, it was measurable in testicular microsomes from rat and mouse, but not monkey or human. No marked species differences in cEH activity were found. In contrast, mEH activity was low in the monkey, intermediate in the rat, and highest in the human and mouse. cGST activity was significantly lower in the two primate species compared with the rat and the mouse. The levels of activity of the xenobiotic metabolizing enzymes studied were generally more than an order of magnitude lower in the testis as compared to the liver. However, in rat and mouse, the levels of mEH and cGST activities in testis were relatively similar to hepatic levels. Overall, these data indicate that species differences in capacity to metabolize xenobiotics may play a role in differential sensitivity to testicular toxicants.

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Egg yolk was spiked with p,p'-dicofol (p,p'-DCF) (0.1-2.0 micrograms/gm), p,p'-dichlorobenzophenone (p,p'-DCBP) (0.1-2.0 micrograms/gm), and 1,1-bis(4-chlorophenyl)-2,2-dichloroethylene (p,p'-DDE) (0.05-1.0 micrograms/gm). The fortified egg yolk (2-5 g) was mixed with acetonitrile to extract non-fat organic materials. After removal of acetonitrile, the spiked chemicals were separated with a column chromatograph packed with acid alumina. Recovery efficiencies for p,p'-DCBP and p,p'-DDE were determined by gas chromatography, and for p,p'-dicofol by high performance liquid chromatography. The recovery efficiencies for p,p'-dicofol, p,p'-DCBP and p,p'-DDE were 77.2-93.8%, 84.1-101.1%, and 88.5-96.0%, respectively.

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Testicular toxicants have become of increasing importance necessitating a better understanding of the possible role of testicular xenobiotic metabolism. The responsiveness of testicular microsomal epoxide hydrolase (mEH), cytosolic epoxide hydrolase (cEH), and cytosolic glutathione S-transferase (cGST) to hepatic inducers was studied in sexually mature male F344 rats and CD-1 mice. The hepatic inducers employed were phenobarbital (PB), beta-naphthoflavone (BNF), and butylated hydroxyanisole (BHA) which are known to induce cytochrome P-450, cytochrome P-448, and cGST, respectively. Hepatic mEH, cEH and cGST activities were assessed as positive controls. Measurable activities of all enzymes studied were present in the testes of both rats and mice. PB, BNF, and BHA produced the expected effects on mEH, cEH, and cGST in rat and mouse livers, whereas the testes were generally nonresponsive to the inducers. Induction of testicular cGST by PB occurred in mice but not rats and was the only testicular effect produced by the hepatic inducers in this study.

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The effect of lindane on 45Ca fluxes in synaptosomes was examined to determine whether modulation of calcium regulation can explain the lindane-induced enhancement in neurotransmitter release observed in other preparations. Lindane (0.03 - 4.6 microM) enhanced calcium uptake in non-depolarizing (low K+) medium: the effect was maximal at 0.1 microM lindane, which increased uptake by 15.8%. Lindane (11.5 microM) decreased calcium uptake in depolarizing (high K+) medium; lower concentrations were ineffective. In addition, lindane (9.2 microM) increased early 45Ca release from synaptosomes. However, lindane (2.4 or 5.9 microM) had no effect on calcium uptake by lysed synaptosomes in the presence or absence of mitochondrial poisons and/or ATP, indicating that lindane does not affect sub-cellular calcium storage. It is concluded that the lindane-induced changes in synaptosomal calcium flux are due to an increase in non-specific synaptic membrane permeability to calcium.

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A definitive hazard assessment of xenobiotics translocated through food animals into edible products such as meat or milk requires a complete analysis of metabolism in food animals. However, large animal metabolism studies present many experimental difficulties. None of several in vitro alternatives such as subcellular fractions has been established as an acceptable predictor of in vivo metabolism. The feasibility of using isolated hepatocytes to predict the metabolism of xenobiotics, both quantitatively and qualitatively, in large ruminant animals (e.g. cattle) is being studied in our laboratory. A procedure was developed for isolating hepatocytes aseptically from the caudate process of the liver which was obtained surgically from 100-125 kg calves. A modified two-step vascular perfusion procedure provides hepatocyte suspensions that are typically greater than or equal to 85% viable and greater than or equal to 1 X 10(7) viable hepatocytes/g of liver (wet wt). Xenobiotic metabolism has been evaluated in suspensions and primary cultures using aldrin epoxidation, ethoxycoumarin O-deethylation, and 7-hydroxycoumarin glucuronidation and sulfation. Metabolic activities are relatively short-lived in suspensions less than or equal to 4 h, but quite stable up to 10 h when cultured on collagen-coated plates in chemically defined medium. Bovine hepatocytes behave similarly in culture to rodent hepatocytes. Although primary culturing of hepatocytes is more difficult than suspensions, primarily due to the asepsis requirements, it is the method of choice for xenobiotic metabolism determinations in isolated hepatocytes of cattle.

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Microsomal and cytosolic enzymes that metabolize xenobiotics were measured in composite samples representing entire livers and in samples from three lobes, using livers of cattle, goats and sheep. Within individual species, concentrations of cytochrome P-450 and b5 and activities of NADPH cytochrome c reductase, aldrin epoxidase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase, microsomal and cytosolic stilbene oxide (epoxide) hydrolase and glutathione S-transferase were not different (P greater than .05) among the various hepatic lobes. Among species, several activities differed (P less than .05), with cattle livers generally having lower values than sheep or goats.

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Young female pastel mink and young female Sprague-Dawley rats were injected intraperitoneally on 3 sequential days with 50 mg/kg of either 2,4,2',4' tetrachlorobiphenyl (TCB) or 3,4,3',4'-TCB and sacrificed after 7 days. Two control groups were established for each species; one allowed free access to food, and one pair-fed to the 3,4,3',4'-TCB-treatment group. Heart blood was collected from each mink immediately after sacrifice. A complete set of tissues was collected from all animals and placed in buffered formalin. The rats displayed no clinical signs of illness following the administration of either congener, nor were there any significant gross or microscopic lesions created in this species. Mink in the 2,4,2',4'-TCB and control groups remained free of clinical signs and significant gross or microscopic lesions. Mink in the 3,4,3',4'-TCB group developed anorexia within 48 hr after the initial injection, and depression and melena by Day 4. Necropsy on Day 7 revealed a severe necrotizing enteritis with moderate to marked villus atrophy and fusion in the small intestines of all mink in this treatment group. The epithelial necrosis generally spared the basal one-third of the mucosa, and the deep crypt epithelium was often moderately hyperplastic. The mechanism by which 3,4,3',4'-TCB causes this unique lesion is unknown.

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Mink have been shown previously to be extraordinarily sensitive to polychlorinated biphenyls (PCBs) and related classes of halogenated hydrocarbons. This study explored several aspects of the acute response of mink to two purified tetrachlorobiphenyl (TCB) congeners and compared their response with that of the rat, a less sensitive and more thoroughly studied species. Young female pastel mink and young female Sprague-Dawley rats received three daily intraperitoneal injections with equimolar doses of either 2,4,2',4'-TCB or 3,4,3',4'-TCB, and were sacrificed after 7 days. Two control groups were used for each species; one was allowed free access to food and the other was pair-fed to the 3,4,3',4'-TCB treatment group. Rats remained clinically normal, while mink treated with 3,4,3',4'-TCB developed severe anorexia, diarrhea, and melena. Both species had significant increases in hepatic cytochrome P-450 content and the characteristic shift in the spectral maxima from 450 to 448 nm in the 3,4,3',4'-TCB- but not in the 2,4,2',4'-TCB-treated animals. Rats but not mink had increased activities of several hepatic monooxygenases in response to both congeners while microsomal epoxide hydrolase was increased in rats after 2,4,2',4'-TCB and in mink after 3,4,3',4'-TCB. Significant increases in the relative volume of smooth endoplasmic reticulum within hepatocytes of 2,4,2',4'-TCB-treated rats but not mink were confirmed by ultrastructural morphometry. Accumulation of both congeners was greater in adipose tissue than in the liver of either species. In both species, concentrations in adipose tissue were much greater for 2,4,2',4'-TCB than for 3,4,3',4'-TCB. PCB toxicosis in mink, as in other species, appeared to be dependent on isomeric arrangement of chlorine substituents. However, unlike other species, the toxicosis was not associated with biochemical or morphological evidence of hepatic enzyme induction. Moreover, the target organ of 3,4,3',4'-TCB toxicosis in mink was the small intestinal mucosa.

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1. The capacity of liver and lung tissue of black-tailed dear (Odocoileus hemionus columbianus) to biotransform xenobiotics was compared in vitro to the domestic sheep and goat. Donor animals were all females of varying ages. Tissues from the black-tailed deer were collected in the wild. A variety of biotransformation enzymes were measured in both microsomal and cytosolic fractions. 2. Deer liver was lower in total cytochrome P450 concentration, but mono-oxygenase activities were greater compared to sheep and goat. The opposite was true for the lung. 3. Epoxide hydrolase activities were significantly different in deer vs sheep and goat. 4. In general, both hepatic and pulmonary activities were more similar between sheep and goat than either species compared to the deer, however, the magnitude of the hepatic differences did not exceed 5-fold. 5. Based on these limited results, there is no reason to discredit the sheep or goat as a toxicity testing model for deer.

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Isolated hepatocytes were prepared from 100- to 125-kg Holstein male calves (n = 10) by perfusion of the caudate process of the caudate lobe of the liver. The 11th or 12th rib on the right side was resected to provide exposure of the caudate process. Complete postsurgical recovery of the donor from partial lobectomy was confirmed by growth data and serum chemical and hematologic criteria. Hepatocytes were isolated under aseptic conditions, using a 2-step collagenase vascular perfusion procedure. Hepatocyte preparations averaged 85% viability, and the yield averaged 1.2 X 10(7) viable hepatocytes/g of (wet weight) liver. Morphologic characteristics of hepatocytes examined under light and scanning electron microscopy were considered normal, except for occasional surface blebs. Freshly isolated hepatocytes in suspension rapidly decreased in viability and xenobiotic metabolizing capacity (aldrin epoxidation and ethoxycoumarin 0-deethylation and 7-hydroxycoumarin glucuronidation and sulfation), and hepatocytes surviving the initial 2 to 3 hours appeared to undergo repair. As an alternative, primary monolayer cultures on collagen-coated plates were evaluated. Hepatocytes attached to the collagen surface within 4 hours and appeared flattened by 12 hours. Although metabolic activity decreased about 30% over 8 hours in culture, the pattern of ethoxycoumarin metabolites was relatively constant. It was not determined to what extent the apparent loss of metabolic capacity was caused by hepatocyte detachment from the collagen surface. Although complicated by the requirement for asepsis, primary cultures were superior to suspensions for xenobiotic metabolism studies in cattle.

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Newborn Holstein bull calves were fed either analytical pentachlorophenol (aPCP) or technical pentachlorophenol (tPCP) for 6 wk to establish and compare the clinical and pathologic manifestations of toxicity. Four groups of three calves/group were each fed either 1 or 10 mg X (kg body weight)-1 X d-1 of either aPCP or tPCP. A fifth group served as control. Dosages of both PCP preparations were normalized to contain equal concentrations of PCP. Toxic effects were observed only at the 10 mg/kg dose in the tPCP-treated calves. These effects included decreased body weight gain, anorexia, decreased serum protein concentration, elevated serum gamma glutamyl transferase, and decreased triiodothyronine (T3) and thyroxine (T4) concentrations. Histologic lesions included cortical atrophy in the thymus and squamous metaplasia and hyperkeratous changes in the Meibomian gland of the eyelid. Thyroid function, which was assessed in vivo by measuring the rate of T3 and T4 production over 4 h after thyrotropin-releasing hormone (TRH)-challenge, was not impaired suggesting an extrathyroidal site of toxic action. Although serum chemistry indicators were suggestive of hepatic injury there were no discernable lesions. Organ weight analyses were inconclusive but there was a tendency toward enlargement of liver, kidneys and thyroid and decreased weight of lungs, spleen and thymus. A toxic effect clearly related to PCP and not its contaminants was depressed active transport of p-aminohippurate measured in kidney slices in vitro. Steady state concentrations of PCP in serum were about 40 and 90 ppm for the 1 and 10 mg/kg groups, respectively. Concentrations of PCP among the major organs were comparable.

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A technique for preparing viable and functional isolated hepatocytes from cattle liver is described. The basic procedure, which was adapted from published methods established for laboratory species, employed a two-step in vitro vascular perfusion of the caudate lobe: (1) perfusion with a calcium-free buffer containing ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA) for removal of blood cells and extracellular calcium and (2) perfusion with calcium-fortified buffer containing collagenase for cell dissociation. Hepatocyte suspensions prepared from the caudate lobes of 20 cattle possessed a mean viability of 81.3% as determined by trypan blue exclusion. Mean yield was 2.2 X 10(7) viable hepatocytes/g of liver (wet wt). Viable hepatocytes utilized O2 at a rate 2.82 times greater than nonviable hepatocytes. Biochemical function of the hepatocyte suspensions was assessed by rates of gluconeogenesis and fatty acid oxidation. Glucose production from added lactate ranged from .88 to 1.47 mumol X min-1 X g-1 of liver tissue (dry wt). Both gluconeogenic and fatty acid oxidation rates were substantially greater in isolated hepatocytes when compared with liver slices. Isolated hepatocyte contained .398 +/- .033 (SE) nmol cytochromes P-450/mg microsomal protein and .285 +/- .025 nmol cytochrome bs/mg microsomal protein, which was comparable with amounts in liver tissue from the same animals (.568 +/- .056 and .298 +/- .033 nmol/mg protein, respectively). No significant decline of either cytochrome was detectable for isolated hepatocytes for up to 5.5 h after euthanasia. The potential usefulness of isolated bovine hepatocytes in xenobiotic metabolism studies is illustrated by the epoxidation of aldrin.

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Mink were injected (ip) daily with 20 mg/kg of 3-methylcholanthrene (MC) or 40 mg/kg of phenobarbital (PB) for 3 days and killed 48 hr after the last injection. The duration of anesthetic action of PB increased after each injection. MC-treated mink became anorexic and lost substantial body weight. PB caused enlargement of liver and lungs, whereas MC caused liver atrophy. No major treatment-related morphologic changes including amount of endoplasmic reticulum (ER) in liver were revealed by electron microscopic examination. Microsomal protein content was not increased and NADPH cytochrome P-450 reductase was not induced in liver by either PB or MC. Cytochrome P-450 (448) was increased 3.2-fold by PB and 2.5-fold by MC. Cytochrome b5 was increased 2.3-fold by MC but was not affected by PB. Aminopyrine N-demethylase was enhanced 5.1-fold in activity by PB whereas hexobarbital hydroxylase was not induced. MC-treatment moderately increased the activities of benzo(a)pyrene hydroxylase (1.7-fold) and ethoxyresorufin O-deethylase (2.1-fold) but had no effect on ethoxycoumarin O-deethylase. The most distinctive features of the mink revealed by this study are a) lack of PB induction of the ER, microsomal protein content, NADPH-cytochrome P-450 reductase, and hexobarbital hydroxylase, and b) lack of MC induction of cytochrome P-448-associated mixed function oxidases that are known to be highly responsive to MC in other species.

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The sensitivity of three genetic lines of Japanese quail to polybrominated biphenyls (PBBs) was evaluated using criteria of egg production, reproduction, and induction of the hepatic microsomal mixed-function oxidase (MFO) system. Two genetic lines of quail, developed to diverge in their plasma cholesterol response to exogenous adrenocorticotropin (ACTH) (a "Low" line and a "High" line), were compared to a random-bred line ("Random"). ACTH administration caused increases in plasma cholesterol in the Low line that were 15 and 39% below the Random-line values in males and females, respectively, while High-line values were 31% higher in males and 36% higher in females when compared to the respective Random-line values. Hepatic activities of aryl hydrocarbon hydroxylase (AHH) and hexobarbital hydroxylase (HxH) were not significantly influenced by ACTH administration or by genetic line in either sex. PBBs fed at 40 or 80 mg/kg diet for 5 wk resulted in significant increases in hepatic AHH and aminopyrine N-demethylase (APND) activities and cytochrome P-450 concentrations. The induction of AHH, APND, and cytochrome P-450 was significantly less in Low-line males in comparison to Random- and High-line males, while the induction of AHH was less in Low-line females when compared to females from the other two lines, based on covariance analysis. In terms of reproductive parameters, there was a greater adverse effect on egg production at 80 ppm PBBs in Low-line females when compared to the Random and High lines. These data indicate an example in which the biological toxicity of a compound and the induction of a 3-methylcholanthrene-type hepatic enzyme are not directly correlated.

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Synthetic and natural toxicants are constituents of soil, air, water and foodstuffs. Their impact on animal agriculture has resulted from acute and chronic intoxication and residues transferred into meat, dairy and poultry products. Recent advances in analytical chemistry and the sciences associated with toxicology have allowed better assessment of the hazard of toxicants on animals including man. Historically, natural toxicants (phytotoxins, mycotoxins and minerals) that are associated with many common feedstuffs accounted for toxicity episodes of epidemic proportions. Most synthetic chemicals (pesticides, nonpesticidal organic chemicals and drugs) have been introduced in increasing numbers since the 1940's. In the 1960's and '70's, recognition of the need to control their environmental distribution stimulated the introduction of numerous laws and regulations. In the last decade, several problematic synthetic chemicals have been banned, particularly those found to persist in the environment or those confirmed or suspected as carcinogens in humans. At the farm level, the development of various preventative management strategies has decreased the exposure of livestock to natural toxicants. In the future, the impact of natural toxicants on animal agriculture is expected to lessen as their existence, etiology and toxicology are determined. On the other hand, synthetic chemicals will continue to threaten animal health as greater numbers and quantities are released into the environment. These challenges should stimulate a greater involvement of animal scientists in toxicology.

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Eight lactating Holstein-Friesian dairy cattle were randomly allotted as pairs to eight a control or a treatment group fed technical pentachlorophenol (penta) from 6 +/-1 wk postpartum for about 135 d (0.2 mg/kg.d for 75-84 d followed by 2.0 mg/kg.d for 56-62 d). Jugular blood was drawn periodically for immunologic studies. Leukocyte differentials and lymphocyte subpopulations (e.g., T and B cells) were enumerated for each blood specimen. Several in vitro and in vivo assays were conducted to evaluate lymphocyte functions, including (1) quantitation of serum immunoglobulins G, M, and A; (2) induction of blastogenesis in vitro, using concanavalin A and leukoagglutinin as mitogens; (3) measurement of extent of skin reaction to injected purified protein derivative in BCG-sensitized cattle to evaluate delayed hypersensitivity; and (4) quantitation of antibody formation in response to injected sheep red blood cells. Neutrophil function was evaluated by latex particle phagocytosis and chemiluminescence. The results showed no statistically significant differences between control and pent-treated cattle during eigher treatment period. Also, no histopathologic changes were noted in lymphoid tissues including spleen, thymus, and lymph node.

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