Biomedical subjects
Publications and source records attributed to D E Moody.
A polymorphism was identified in the bovine PIT1 gene by digesting polymerase chain reaction (PCR) products with the restriction enzyme HinfI. This polymorphism was segregating in five diverse breeds of cattle. PIT1 was sublocalized to the centromeric region of bovine chromosome 1 by linkage analysis.
A polymorphism was identified in the bovine growth hormone receptor (GHR) gene by digesting polymerase chain reaction (PCR) products with the restriction enzyme Alul. Two alleles were segregating in cattle of Bos indicus descent, but one allele appears to be fixed in Bos taurus cattle. GHR was localized to bovine chromosome 20 using bovine-rodent hybrid cell lines and linkage analysis.
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Urinary excretion of amphetamine and 4'-hydroxyamphetamine has been studied in male and female Sprague Dawley (SD) and Dark Agouti (DA) rats. The DA rat is an animal model for the cytochrome P450 (P450) 2D poor metabolizer. Rats were given d-amphetamine sulfate (5 mg/kg, i. p.) and urines were collected at 12 hour intervals for extraction and analysis of the amphetamines by HPLC. There was no significant difference between the sexes of either SD and DA rats in urinary 4'-hydroxyamphetamine and amphetamine excretion, but significant differences were seen between the two strains. The percentage of dose per ml urine recovered as 4'-hydroxyamphetamine from the urine over 24 hours was 11.1 and 9.1 in the SD male and female rats, and 2.3 and 2.5 in DA male and female rats, respectively. The percentage of dose per ml urine recovered as amphetamine was correspondingly lower in the SD male and female rats, 1.1 and 1.0, than that of the DA male and female rats, 5.9 and 5.0. These results support our hypothesis that P450 2D is involved in hepatic 4'-hydroxylation of amphetamine in rats.
The ability of commercial benzodiazepine immunoassays to detect nordiazepam, lorazepam, alprazolam, and triazolam in blood samples was investigated. Two radioimmunoassays (RIA) (Abuscreen [RIA-a] and the Diagnostic Products Corporation serum kit [RIA-d]), two enzyme immunoassays (EIA) (Emit d.a.u. [EIA-u] and Emit TOX serum assay [EIA-s]), and two fluorescence polarization immunoassays (FPIA) (X-systems urine [FPIA-u] and X-systems serum assay [FPIA-s]) were evaluated for their ability to detect benzodiazepines in fortified drug-free human or bovine blood. Prior extraction of the blood was necessary for analysis on the equipment used for EIA and FPIA. Extraction with an organic solvent, such as butyl chloride used in this study, was preferable to precipitation with methanol or zinc sulfate. For all these assays, extraction eliminated matrix effects and offered the possibility of increased sensitivity by reconstitution of the extract in a smaller volume. Extraction was necessary in bovine blood, as there is some nonextractable substance(s) in this matrix that increases non-specific binding. Using extraction with reconstitution in one-half the original volume, the apparent limit of detection for nordiazepam in blood ranged from 3 ng/mL, with RIA-d, to 30 ng/mL, with EIA-s and FPIA-s. These limits of detection were improved by further reduction of the reconstitution volume. RIA-a, EIA-s, and FPIA-s had cross-reactivity for alprazolam that was equivalent to or slightly better than nordiazepam. RIA-d had enhanced cross-reactivity for alprazolam at 30-300 ng/mL.(ABSTRACT TRUNCATED AT 250 WORDS)
Debrisoquine and sparteine are prototype substrates of a genetic deficiency in cytochrome P450-dependent drug metabolism. Sensitive assays of in vitro oxidation of sparteine and debrisoquine are required for evaluation of this polymorphism. The activities were measured by quantitative analysis of 2-dehydrosparteine and 4-hydroxydebrisoquine production, respectively, using capillary column gas chromatography coupled with mass selective ion detection. With a single extraction, separation of parent drug, metabolite, and a suitable internal standard was readily achievable. Time-dependent production of both metabolites could be detected from as little as 40 micrograms of microsomal protein. Both activities showed a maximal activity with a 240-min incubation period. The ability to simultaneously quantify the parent drug and its metabolite suggests it would also be useful for evaluation of in vivo metabolism.
The effect of phenobarbital treatment on the linkage between carbon tetrachloride-mediated cytochrome P-450 loss and lipid peroxidation in rat liver microsomes was studied. Male Sprague-Dawley rats, pretreated with 3 daily i.p. doses of phenobarbital (50 mg/kg) or saline, were orally dosed with carbon tetrachloride (0.01-2.5 ml/kg), with liver microsomes prepared at 7.5-180 min after carbon tetrachloride treatment. In vivo cytochrome P-450 loss displayed pseudo-first-order kinetics, and the initial rates of diene conjugate formation were saturable with dose. Phenobarbital pretreatment decreased the in vivo t0.5,max from 27.0 to 15.6 min, and increased the Kd,app from 0.78 to 1.30 ml/kg for carbon tetrachloride mediated cytochrome P-450 loss. Phenobarbital had no effect on the in vivo Vmax (1.03 to 1.04 delta OD232 nm/min/mg phospholipid) for carbon tetrachloride mediated diene conjugate formation, but decreased the Km,app from 0.22 to 0.10 ml/kg. These results are consistent with destruction of cytochrome P-450 heme resulting from a metabolite which does not leave the site of generation, and with phenobarbital pretreatment enhancing the initiation of lipid peroxidation.
Milk yield of 59 beef cows that calved in late September through November was measured monthly in early and late lactation and biweekly during midlactation. Milk yield was estimated by milking with a machine after over-night separation of cows from calves. Concentration of plasma cholesterol of cows and calves was measured when calves averaged 44, 93, 136, and 178 d of age (SD = 17 d). Cholesterol of calves also was measured 2 wk after weaning, when they averaged 220 +/- 2.3 d of age. Cholesterol of calves was highest at second and third samplings and dropped after weaning. The estimated intake of milkfat by calves, and to a lesser extent their intake of milk, was related positively to their plasma cholesterol as they approached weaning age. The relationship was not strong enough, however, for differences among calves in concentration of plasma cholesterol to identify accurately differences in milk yield of their dams. Within breed group, age of cow, and stage of lactation, the regression coefficients of milk yield on plasma cholesterol of cows were close to zero. The concentrations of plasma cholesterol in both cows and calves were highly repeatable, with the exception of samples that were collected when calves averaged 44 d of age. Although plasma cholesterol of calves was related positively to milk yield and milkfat yield in late lactation, the former trait was not an accurate indicator of the two latter traits.
Male mice were treated (i.p.) for 3 days with 15 different environmentally encountered epoxides, and the effects of these compounds on liver microsomal and cytosolic epoxide hydrolase (mEH and cEH), glutathione S-transferase (mGST and cGST) and carboxylesterase (mCE) activities were determined. The epoxides included the pesticides: heptachlor epoxide, dieldrin, tridiphane, and juvenoid R-20458; the natural products: disparlure, limonin, nomilin, and epoxymethyloleate; the endogenous steroids: lanosterol epoxide, cholesterol-alpha-epoxide, and progesterone epoxide; and the industrial or synthetic epoxides: epichlorohydrin, araldite, trans-stilbene oxide, and 4'-phenylchalcone oxide. The pesticide epoxides were the most effective inducers of liver weight, microsomal protein, and the enzyme activities measured, with mEH and cEH activities towards cis-stilbene oxide (mEHcso and cEHcso), cGST activities towards four of five substrates, and mCE towards clofibrate (mCEclof) and p-nitrophenylacetate (mCEpna) increased following treatment with most of the pesticides. The synthetic epoxides increased some of the same activities, while the natural products, except for increases in cGST activities, and endogenous steroid epoxides were generally not inductive. cEH activity towards trans-stilbene oxide (cEHtso) was increased only following treatment with the peroxisome proliferator, tridiphane, but decreased following treatment with several of the epoxides, while microsomal cholesterol epoxide hydrolase (mEHchol) was increased only moderately by disparlure. Microsomes could effectively conjugate glutathione to chlorodinitrobenzene (mGSTcdnb) and cis-stilbene oxide (mGSTcso). These two activities were differentially induced by a few of the epoxides, suggesting that they may be selective substrates for different isozymes of mGST. Correlation coefficients were determined for the relative response of liver weight, subfraction protein, and enzyme activities. A relatively high correlation was found between the response of liver weight and cytosolic hydrolysis of trans-stilbene oxide (r = 0.73) and cis-stilbene oxide (r = 0.62), and cytosolic glutathione conjugation of dichloronitrobenzene (r = 0.66) and trans-stilbene oxide (r = 0.75). In addition, relatively high correlations were found between the different cGST activities, in particular for dichloronitrobenzene with trans-stilbene oxide (r = 0.89). These studies show that there exists a wide variation in the response of xenobiotic-metabolizing enzymes to environmentally encountered epoxides and that a fairly strong correlation exists between the increases in liver size and increases in certain cytosolic enzyme activities; they also suggest further studies concerning the possibility of an additional isozyme of mGST.
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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.
Tetrahydrofuran (THF) is a widely used solvent in industry and research. THF is a weak toxin, with approximate acute LD50s in the range of 2 to 3 g/kg, 8 to 20 mg/L, and 800 mg/kg following oral, inhalation, and i.v. routes, respectively. The two primary signs of intoxication are narcosis and hepatocellular dysfunction, both occurring at doses of approximately one-half of the lethal dose. Little is known concerning the pharmacokinetics of THF. No evidence exists for genotoxicity due to THF. Ongoing carcinogenicity bioassay tests sponsored by the National Toxicology Program have not yet been completed. Two significant interactions of THF with cellular components have been studied; first, THF inhibits a number of enzymatic reactions at concentrations ranging from 10 to 100 mM. Most notably, THF is an inhibitor of a number of cytochrome P450 (P450) dependent mixed function oxidase activities, with a particular affinity for the alcohol-induced isozyme (P450IIE1). Second, THF has been noted to enhance the toxic action of a number of compounds (i.e. solvent effect), in particular stimulating the more rapid absorption of reactive metabolites. A third factor to be considered is THF's ability to form peroxides upon standing. Little is known concerning the toxicity of THF peroxides. Therefore, while there is little evidence to suggest that THF would be a direct (Type I) hepatotoxin at relatively low doses, its ability to inhibit drug-metabolizing reactions (perhaps more so in alcohol users), and enhance the absorbance of reactive metabolites, are relatively unexplored avenues for THF to contribute to a hepatotoxic response. As a great deal of the human hepatotoxicity (Type II) is "idiosyncratic" and cannot be readily reproduced in experimental animals, this potential route of hepatotoxicity should be considered in future evaluations of human exposure to THF.
Postaccident testing of railroad employees for drugs and alcohol was performed in 175 qualifying accidents or incidents (events) from April 1987 through March 1988. Initial tests for drugs were performed on urine, and for alcohol on blood. Presumptive positives were confirmed and quantitated using both blood and urine when available. In 42 of these events (24%), at least 1 employee tested positive (6.7% of 736 employees) for drugs or alcohol. A higher proportion of drug or alcohol-positive events, or both, 40 versus 21%, was found when a fatality was involved. In 11 of the 32 fully investigated drug or alcohol-positive events (involving 14 employees), the investigating agencies determined that substance use was determined to be a probable cause of, or a factor relating to, the accident. Cannabinoids, ethanol, cocaine, or multiple-drug use were found in 5, 3, 3, and 3 of those employees, respectively. Detectable drug and alcohol use occurs among railroad employees; occasionally it has resulted in accidents.
Phenobarbital treatment induces an isozyme(s) of liver microsomal cytochrome P450 susceptible to CCl4 and enhances the latter's lethality. We have now studied phenobarbital's effect on the specificity of phosphatidyl fatty acid changes in rat liver microsomes. Male Sprague-Dawley rats were pretreated with three daily ip doses of phenobarbital (50 mg/kg) or saline and then orally dosed with CCl4 (2.5 ml/kg). Liver microsomes were prepared 7.5 to 180 min after CCl4 treatment, the lipid fraction was extracted, diene conjugate content was determined, and phospholipids were separated by HPLC for fatty acid content determination. Protein, phospholipid, and phosphatidyl fatty acid residue loss occurred early (7.5 to 30 min) and in some cases later (60 to 180 min) in both pretreated groups, suggesting that two phases of CCl4-mediated injury occurred. Phenobarbital pretreatment accelerated the CCl4-induced formation of diene conjugates in the microsomal lipids. In studies on the separated phospholipids, phenobarbital alone altered microsomal fatty acid content, primarily decreasing arachidonic acid in favor of linoleate, particularly in phosphatidylserine. During the early phase of CCl4 injury, phenobarbital pretreatment shifted the major loss of arachidonic acid from phosphatidylserine to phosphatidylethanolamine. During the later phase, arachidonic acid loss was still prominent, but the most extensive CCl4-induced changes in fatty acids occurred in the neutral lipid fraction, regardless of pretreatment. These changes included loss of neutral lipid linoleic and docosahexanoic acids associated with an increase in palmitic acid. These data demonstrate that phenobarbital pretreatment is associated with a shift in the predominant phospholipid locus from phosphatidylserine to phosphatidylethanolamine for the early CCl4-induced fatty acid changes in rat liver microsomes.
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An antibody (anti-EH) specific for microsomal epoxide hydrolase (mEH) from rhesus monkey liver has been used to test the immunochemical relationship between human liver mEH and the serum EH levels in human patients with hepatocellular carcinoma (HCC). Immunoblots of separated rhesus monkey and human liver microsomal proteins revealed that anti-EH was selective for a single polypeptide band of similar mol. wt, approximately 49 kd, in both species. Anti-EH was also able to precipitate 100% of the activity for two substrates specific in the mouse for mEH, cis-stilbene oxide and benzo[a]-pyrene-4,5-oxide, in solubilized human liver microsomes. In contrast, only 20% of the microsomal trans-stilbene oxide hydrolase activity was precipitated under similar conditions, providing immunochemical evidence that a distinct EH, with substrate selectivity similar to the cytosolic EH, resides in human liver microsomes. Immunoprecipitation of serum from a patient with elevated EH activity resulted in total precipitation of cis-stilbene oxide hydrolase activity. An enzyme-linked immunoabsorbant assay (ELISA) was developed using anti-EH with detection limits of 1 ng/ml. A high correlation between the enzymatically and immunochemically determined levels of serum EH provided further evidence for the immunochemical similarity of human liver microsomal and serum EH. In addition, the ELISA was equally capable of identifying elevated serum EH in patients with HCC, and should prove invaluable in evaluating the effectiveness of serum EH levels as a marker for HCC.
DNA is the purported target of several carcinogenic and mutagenic agents. Nuclear enzymes which could generate or detoxify reactive metabolites are of major concern. Several such enzymes have been identified within nuclei, but obtaining samples with enriched content or activity is difficult, time-consuming, and uses harsh isolation techniques. Extraction of rat liver nuclear suspensions with cholate-containing buffer results in solubilization of 25-30% of the protein. Linear extraction was obtained for total protein and cytochromes P-450 and b5, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, DT-diaphorase, and microsomal-like epoxide hydrolase with specific activities comparable to values reported for isolated nuclear membrane, while the yield was five to ten times greater. Detergent extracts of rat liver nuclei were employed to study the comparative response of microsomal and nuclear enzymes to chemical treatment. While the responses to acute inductive (phenobarbital and 3-methylcholanthrene) and toxic (carbon tetrachloride and dibromochloropropane) treatments were qualitatively similar, an initiation-promotion protocol (diethylnitrosamine with phenobarbital promotion) resulted in divergent responses between the enzymes in the two subcellular fractions. Detergent extracts of nuclei offer an efficient means of recovering xenobiotic-metabolizing enzymes from rat liver nuclei, and have been utilized to demonstrate a differential response of nuclear enzymes during preneoplastic development.
Rat liver sections were incubated with antibodies (100-1000 micrograms IgG/ml) against microsomal cytochromes P-450a, P-450b, and P-450c, and epoxide hydrolase. Inhibition of indirect immunofluorescence, which progressed with higher concentrations of primary antibody, corresponded with antigen-enriched tissue in frozen liver sections from male and female rats. It was found in liver sections from phenobarbital-treated rats incubated with anti-P-450b and anti-epoxide hydrolase and from 3-methylcholanthrene-treated rats incubated with anti-P-450c. No inhibition was found in sections from untreated rats or rats receiving treatments that did not induce the specific antigen. No inhibition was found in sections incubated with anti-P-450a. Inhibition of immunofluorescence was abolished in frozen sections subjected to dehydration-rehydration protocols known to extract antigens, and was prevented by certain solvents and detergent-wash. Inhibition of immunofluorescence provides a unique method for confirming the antigen-rich regions of the liver lobules specific for microsomal expoxide hydrolase and the cytochrome P-450s.