Morphometric analysis of the ultrastructural changes in rat liver induced by the peroxisome proliferator SaH 42-348.

The changes occurring in hepatocytes of F-344 male rats during a 3-wk treatment with a hypolipidemic agent, 1-methyl-4-piperidyl-bis [p-chlorophenoxy]acetate (SaH 42-348), have been evaluated by morphometric and biochemical methods. The twofold increase in liver weight resulted from a significant increase in hepatocyte cytoplasm as well as a moderate increase in the number of liver cells. The peroxisome population and SER played an overwhelming part in the hypertrophy of hepatocytic cytoplasm. The relative volume and the surface density of peroxisomes volume resulted from an increased ninefold and sevenfold, respectively. The increase in the collective peroxisome volume resulted from an increase in both the number and the average volume of peroxisomes. The SER also demonstrated a substantial increase in these values. The relative volume and surface density of mitochondria were not significantly altered in comparison to controls, while these values for RER decreased onefold. Studies on the lobular distribution of cytoplasmic organelles before and during treatment revealed that the relative volume and surface density of peroxisomes and SER increased from periportal to centrilobular cells of the hepatic lobule, whereas mitochondrial values decreased from periportal to centrilobular cells. The RER values were fairly constant in different parts of the hepatic lobule. The increase in peroxisome and SER volume and surface area was first evident within the first 3 days of SaH 42-348 treatment and these values continued to increase, reaching a steady state within 2 wk. The time course of increase in catalase and carnitine acetyltransferase activities correlated with the morphometric data on the peroxisomes. After cessation of SaH 42-348 treatment, the peroxisome values decreased rapidly within the first 3 days and reached control levels within 1 wk. Moderate reduction in SER values occurred after withdrawal of the drug, but these values remained higher than controls even after 2 wk, suggesting that the reduction in the amount of circulating peroxisome proteins may result in empty SER channels. On the 4th day of drug withdrawal a significant increase in the relative volume and surface density of lysosomes was observed, suggesting that these organelles may play some part in the removal of cellular membranes. However, the rapid reduction in peroxisome values after SaH 42-348 withdrawal appears to be due to cessation of enhanced peroxisome protein synthesis.

Hepatocellular carcinomas in acatalasemic mice treated with nafenopin, a hypolipidemic peroxisome proliferator.

The effects of long-term administration of nafenopin, a potent hypolipidemic drug with marked hepatomegalic and peroxisome-proliferative properties, were studied in wild-type (Csa strain) and acatalasemic (Csb strain) mice. Nafenopin was administered in the diet at a concentration of 0.1% during the first 12 months and then at 0.05% until the termination of the experiment at 20 months. By 56 weeks, 100% mortality occurred in both male and female wild-type mice, whereas the mortality rate in acatalasemic mice was approximately 50%. Between 18 and 20 months of the experiment, 9 of 9 male and 12 of 12 female acatalasemic mice that survived chronic nafenopin treatment developed hepatocellular carcinomas, some of which metastasized to the lungs. None of the 15 male and 15 female acatalasemic controls developed liver cancers. Numerous peroxisomes were seen in the lung metastases of these hepatocellular carcinomas on electron microscopic examination; in contrast the number of peroxisomes in primary liver tumor cells varied considerably. The hepatocarcinogenicity of nafenopin strongly suggests the need for long-term studies with other hypolipidemic drugs that cause hepatomegaly and peroxisome proliferation to clarify the role, if any, of peroxisome proliferation in liver carcinogenesis.

Effects of thioacetamide treatment on nuclear envelope nucleoside triphosphatase activity and transport of RNA from rat liver nuclei.

An in vitro system was used for investigating transport of rapidly labeled RNA from liver nuclei isolated from control and thioacetamide-treated rats. An enhanced transport was observed in preparations from thioacetamide-treated animals. Analysis of transport at various temperatures indicated that it proceeded as an energy-requiring process, even in the absence of an exogenously added energy source, and was related to hydrolysis of high-energy nucleoside triphosphate esters. In control preparations, a nucleoside triphosphatase (NTPase) is present in nuclear envelopes, and this TNPase activity appears to be intimately related to in vitro RNA transport. Nuclear membranes were isolated from purified nuclear preparations taken from control and thioacetamide-treated rats and were examined for NPTase activity. The NTPase activity was significantly greater in the preparations from thioacetamide-treated animals. These observations indicate that an early change associated with carcinogen exposure is an enhanced NTPase activity. This may underlie the enhanced RNA transport observed in vitro.

Apparent differential response of nuclear envelope cytochrome P-450 following phenobarbital induction arising from a preferential loss during gradient purification.

We have investigated the response of rat liver nuclear, nuclear envelope, and microsomal cytochrome P-450 (or P-448) to various treatments. Responses of these subcellular fractions to 3-methylcholanthrene pretreatment were generally similar. In endoplasmic reticulum preparations, we observed an increase in cytochrome P-450 content following phenobarbital pretreatment, which was reduced by subsequent thioacetamide treatment. Nuclear envelope cytochrome P-450 was apparently not modulated by these treatments, although nuclear cytochrome P-450 content was increased by phenobarbital. When endoplasmic reticulum preparations were subjected to treatments paralleling those used in nuclear envelope purification, we found a preferential loss of cytochrome P-450 from phenobarbital-pretreated preparations, with a loss of camphor-binding ability. The data point to potential problems with use of isolated nuclear envelopes as a representative model for nuclear metabolism of carcinogens, including low total recoveries and enrichments, and the potential for selective or differential recovery of cytochrome P-450 populations following various modes of induction or reduction.

Nuclear envelope alterations accompanying thioacetamide-related enlargement of the nucleus.

Treating rats with thioacetamide results in a biphasic nuclear swelling, with an initial enlargement phase (0 to 8 hr) and a secondary phase (18 to 48 hr). The swelling coincides with and may be responsible for alterations in sedimentation properties of isolated nuclei. Nuclear swelling implies increases in nuclear surface area. Total nuclear lipid increased in proportion to the increase in nuclear-envelope (NE) surface area, suggesting that intranuclear lipid was not the source of new NE phospholipid. The increased NE was apparently not derived from mass incorporation of endoplasmic reticulum into NE, as demonstrated by monitoring enzymatic composition. Significant differences were observed in the distribution of p.o. administered [14C]stearic acid and [14C]phosphatidylcholine in lipid fractions from NE as compared with those from endoplasmic reticulum. Analysis of NE phospholipid revealed little change in composition in the heavy fraction during the first 48 hr after treatment. (An early decrease in phosphatidylserine from the light nuclear fraction may reflect the upward shift of nuclei from the heavy fraction.) A significant increase in phosphatidylserine of NE from the heavy fraction was found at 96 hr. The additional phosphatidylserine in the NE might act as an acidic polymer modifying chromatin structure. Examination of fatty acids from individual NE phospholipid fractions divulged relatively few changes related to the early or the secondary nuclear swelling phases. Major changes occurred in the neutral lipid fraction of NE which apparently served as a reservoir for 18:2, with significant decreases in 18:2 noted following both nuclear swelling phases. These changes did not occur in the neutral lipid fraction from endoplasmic reticulum obtained from the same liver preparations.

Identification of quantitative trait loci influencing traits related to energy balance in selection and inbred lines of mice.

Energy balance is a complex trait with relevance to the study of human obesity and maintenance energy requirements of livestock. The objective of this study was to identify, using unique mouse models, quantitative trait loci (QTL) influencing traits that contribute to variation in energy balance. Two F2 resource populations were created from lines of mice differing in heat loss measured by direct calorimetry as an indicator of energy expenditure. The HB F2 resource population originated from a cross between a noninbred line selected for high heat loss and an inbred line with low heat loss. Evidence for significant QTL influencing heat loss was found on chromosomes 1, 2, 3, and 7. Significant QTL influencing body weight and percentage gonadal fat, brown fat, liver, and heart were also identified. The LH F2 resource population originated from noninbred lines of mice that had undergone divergent selection for heat loss. Chromosomes 1 and 3 were evaluated. The QTL for heat loss identified on chromosome 1 in the HB population was confirmed in the LH population, although the effect was smaller. The presence of a QTL influencing 6-wk weight was also confirmed. Suggestive evidence for additional QTL influencing heat loss, percentage subcutaneous fat, and percentage heart was found for chromosome 1.

Validation of dual-energy X-ray absorptiometry in live White Leghorns.

Dual energy x-ray absorptiometry (DEXA) was evaluated for use as a noninvasive tool to monitor skeletal integrity in live laying hens. The objectives of the current study were 1) to validate the use of DEXA in evaluating bone integrity in live birds as compared with excised bones under a normal nutritional regimen as well as in hens fed varying levels of dietary Ca and 2) to correlate densitometric scans with other bone strength criteria and egg traits. Densitometric scans were conducted on the tibia and humerus of live hens at 10-wk intervals from 17 to 67 wk of age. After each scan, bones were excised from euthanized hens to measure breaking strength characteristics and bone ash (experiment 1). Similar measurements were collected at 38, 48, and 58 wk of age from hens fed hypercalcemic (5.4%), control (3.6%), and hypocalcemic (1.8%) diets from 32 to 58 wk of age (experiment 2). The bone mineral density (BMD) and bone mineral content (BMC) between live and excised bone scans were highly correlated (r = 0.85 and 0.92, respectively, P < 0.0001, experiment 1). Densitometric scans of live birds were positively correlated with bone breaking force and bone ash (r = 0.68 and 0.73, respectively, P < 0.001) with little to no correlation with shell traits. In experiment 2, the excised tibial scan had lower BMD and BMC than the live bird (P < 0.01), whereas no difference was detected in densitometric scans of the humerus. The live and excised BMD and BMC of the tibia (r = 0.87 and 0.82, respectively, P < 0.001) and humerus (r = 0.94 and 0.93, respectively, P < 0.001) were highly correlated. Due to the high correlations between live and excised bone scans and the significant correlations of live scans to more traditional invasive bone measurement tests such as bone breaking force and bone ash, we concluded that DEXA is a useful noninvasive tool for evaluating skeletal integrity in live birds.

Cross-species hybridisation of pig RNA to human nylon microarrays.

BACKGROUND: The objective of this research was to investigate the reproducibility of cross-species microarray hybridisation. Comparisons between same- and cross-species hybridisations were also made. Nine hybridisations between a single pig skeletal muscle RNA sample and three human cDNA nylon microarrays were completed. Three replicate hybridisations of two different amounts of pig RNA, and of human skeletal muscle RNA were completed on three additional microarrays. RESULTS: Reproducibility of microarray hybridisations of pig cDNA to human microarrays was high, as determined by Spearman and Pearson correlation coefficients and a Kappa statistic. Variability among replicate hybridisations was similar for human and pig data, indicating the reproducibility of results were not compromised in cross-species hybridisations. The concordance between data generated from hybridisations using pig and human skeletal muscle RNA was high, further supporting the use of human microarrays for the analysis of gene expression in the pig. No systematic effect of stripping and re-using nylon microarrays was found, and variability across microarrays was minimal. CONCLUSION: The majority of genes generated highly reproducible data in cross-species microarray hybridisations, although approximately 6% were identified as highly variable. Experimental designs that include at least three replicate hybridisations for each experimental treatment will enable the variability of individual genes to be considered appropriately. The use of cross-species microarray analysis looks promising. However, additional validation is needed to determine the specificity of cross-species hybridisations, and the validity of results.

Serum epoxide hydrolase (preneoplastic antigen) in human and experimental liver injury.

Reports of an increase in a serum epoxide hydrolase (sEH), immunochemically related to microsomal EH in humans and rats with hepatocellular carcinoma (HCC), suggested its use as a serum marker for this disease. We have now measured sEH levels (as either immunochemically determined content or enzyme activity) in a number of human and experimental models of liver disease. sEH was elevated above the normal range in at least 50% of individuals with HCC, including: 3 of 6 northern Californians; 4 of 7 Koreans with hepatitis B-associated HCC; hepatitis B-associated HCC in woodchucks; and male rats receiving chronic treatment with aflatoxin B1 or ciprofibrate. sEH was rarely elevated in other forms of chronic liver disease. Only 2 of 9 Koreans with hepatitis B-associated cirrhosis, 1 of 8 carriers, but none with chronic active hepatitis or infection with no apparent liver disease had elevated sEH. In addition, no elevations were found in woodchucks with noncancerous viral hepatitis. In aflatoxin B1- and M1-treated rats sEH was not elevated in those with only hyperplastic foci or hepatocellular adenomas, and in two rat initiation-promotion protocols sEH was elevated only in those rats which received the entire set of treatments. sEH was also increased during acute hepatotoxicity in rats treated with CCl4 or 1,2-dibromo-3-chloropropane. The mechanism of increase in sEH during hepatocarcinogenesis appears to be different from that of other markers of HCC, for in the Korean patients, there was no correlation between sEH concentrations and those of alpha-fetoprotein or ferritin, nor was there a correlation with alpha-fetoprotein concentrations in the aflatoxin-treated rats. Furthermore, the increase in sEH does not correlate with induction of microsomal EH in the liver of experimental animals. Studies to date indicate that sEH is selective for HCC and severe hepatonecrotic injury, and may be of some use in the diagnosis of HCC, particularly as a complement to other serum markers.

Differential inhibition of indirect immunofluorescence in rat liver sections incubated with antibodies against microsomal cytochromes P-450a, b, and c and epoxide hydrolase.

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.

Peroxisome development in the regenerating pars recta (P3 segment) of proximal tubules of the rat kidney.

The development of peroxisomes, lysosomes and endocytic vacuoles in regenerating cells of the pars recta (P3 segment) of proximal tubules, in rats given a single interperitoneal injection of d-serine (80 mg/100 g.b.wt), was studied by light and electron microscopy using cytochemical methods. Rapid proliferation of cells occurred between 2 and 5 days after d-serine induced tubular necrosis; by day 6 almost all injured tubules were re-epithelialized with flat or low cuboidal cells. Peroxisomes and lysosomes were not observed during the period of rapid cell multiplication i.e., between 2 and 6 days after d-serine injection. Restitution of mitochondrial population preceded the development of peroxisomes in the newly regenerated cells of P3 tubules. Maximum development of peroxisomes occurred between 9 and 14 days after d-serine injection. The formation of peroxisomes appeared to correlate closely with the differentiation of apical endocytic vacuoles and the brush border. Lysosomes in the regenerated cells of P3 tubules were the last to develop.

Epoxide hydrolysis in the cytosol of rat liver, kidney, and testis. Measurement in the presence of glutathione and the effect of dietary clofibrate.

The hydrolysis of trans- and cis-stilbene oxide and benzo[a]pyrene-4,5-oxide was measured in cytosol and microsomes of liver, kidney, and testis of control and clofibrate-fed rats. Significant levels of nonprotein sulfhydryls were detected in cytosol from liver (4.6 mM) and testis (1.5 mM). Glutathione was moderately stable in these fractions and interfered with the partition assays as conjugates were retained in the aqueous phase along with diols. When the products were separated by thin-layer chromatography, significant amounts of glutathione-conjugates were found to have been formed in the cytosol of liver and testis. Overnight dialysis or preincubation of cytosol with 0.5 mM diethylmaleate eliminated conjugate formation without affecting diol production. In dialyzed cytosol from clofibrate-fed rats (0.5%, 14 days), the rates of hydrolysis of trans-stilbene oxide were 506, 171, and 96% of controls for liver, kidney, and testis, respectively, and 126% of controls in liver microsomes. Rates of hydrolysis of cis-stilbene oxide were 149, 172, and 96% of controls in microsomes and 154, 124, and 91% of controls in cytosols from livers, kidneys, and testis of clofibrate-fed rats respectively. Hydrolysis of benzo[a]pyrene-4,5-oxide was similar to that of cis-stilbene oxide. Conjugation of the cis-stilbene oxide with glutathione was detected in cytosols from all three tissues with lesser amounts in the microsomes from liver and kidneys. After clofibrate treatment, the rates of this activity were 200, 173, and 95% of controls in cytosol from liver, kidneys and testis, and 203 and 202% of controls in microsomes from liver and kidneys respectively. These results indicate that epoxide hydrolysis and conjugation in rat liver and kidney are responsive to clofibrate treatment and support other evidence which suggests that hydrolysis of cis- and trans-stilbene oxides in cytosol is catalyzed, in part, by distinct enzymes.

Peroxisome-associated enzymes and serum lipids in tumour-bearing rats treated with peroxisome-proliferating agents.

Xenobiotic induction of liver peroxisomes is associated with hypolipidemia. To test the involvement of the peroxisome proliferation with the hypolipidemia, male rats were inoculated in the groin with five different tumors: an aflatoxin-induced hepatoma, a lasiocarpine-induced hepatoma, an actinomycin-D-induced mesothelioma, a lasiocarpine-induced squamous cell carcinoma, and a methylnitrosourea-induced fibrosarcoma. After the tumours reached a suitable size, the rats were fed diets containing the peroxisome-proliferating hypolipidemic agents tibric acid (2-chloro-5-[3,5-dimethylpiperidinosulfonyl] benzoic acid) or Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid) for 2 weeks. Liver and tumor tissues were then assayed for the peroxisome-associated enzymes, catalase and carnitine acetyltransferase, and correlated with serum levels of triglyceride and cholesterol. The presence of the tumors caused a predictable decrease in liver catalase and a slight elevation of liver carnitine acetyltransferase. Serum cholesterol was elevated slightly, while serum triglyceride levels were elevated, unchanged, or decreased in the tumor-bearing rats maintained on control diet. Inclusion of the xenobiotics in the diet caused increases in liver weight, catalase, and carnitine acetyltransferase. Serum triglycerides were decreased in the three groups which were not already decreased, but a decrease in serum cholesterol was only found in one group after only one of the treatments. The latter finding demonstrates that peroxisomal enzyme induction can be dissociated from the decrease in serum cholesterol. The data were further evaluated by testing for correlations between the changes in these components, comparing changes within groups and between groups. These correlations indicate an inverse biological association between liver catalase and serum cholesterol and between liver carnitine acetyltransferase and serum triglyceride. The latter correlation was inverse only for comparisons between groups, suggesting that carnitine acetyltransferase activity is associated with serum triglycerides only during the perturbational state.

Effect of dietary clofibrate on epoxide hydrolase activity in tissues of mice.

The effects of dietary clofibrate on the epoxide-metabolizing enzymes of mouse liver, kidney, lung and testis were evaluated using trans-stilbene oxide as a selective substrate for the cytosolic epoxide hydrolase, cis-stilbene oxide and benzo[a]pyrene 4,5-oxide as substrates for the microsomal form, and cis-stilbene oxide as a substrate for glutathione S-transferase activity. The hydration of trans-stilbene oxide was greatest in liver followed by kidney greater than lung greater than testis. Its hydrolysis was increased significantly in the cytosolic fraction of liver and kidney of clofibrate-treated mice and in the microsomes from the liver. Isoelectric focusing indicates that the same enzyme is responsible for hydrolysis of trans-stilbene oxide in normal and induced liver and kidney. Clofibrate induced glutathione S-transferase activity on cis-stilbene oxide only in the liver. Hydrolysis of both cis-stilbene oxide and benzo[a]pyrene 4,5-oxide was highest in testis followed by liver greater than lung greater than kidney. Hydration of cis-stilbene oxide was induced significantly in both liver and kidney by clofibrate but that of benzo[a]pyrene 4,5-oxide was induced only in the liver. These and other data based on ratios of hydration of benzo[a]pyrene 4,5-oxide to cis-stilbene oxide in tissues of normal and induced animals indicate that there are one or more novel epoxide hydrolase activities which cannot be accounted for by either the classical cytosolic or microsomal hydrolases. These effects are notable in the microsomes of kidney and especially in the cytosol of testis.

Effects of environmentally encountered epoxides on mouse liver epoxide-metabolizing enzymes.

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.

Sodium cholate extraction of rat liver nuclear xenobiotic-metabolizing enzymes.

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.

Urinary excretion of amphetamine and 4'-hydroxyamphetamine by Sprague Dawley and dark Agouti rats.

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.

Determination of tolbutamide hydroxylation in rat liver microsomes by high-performance liquid chromatography: effect of psychoactive drugs on in vitro activity.

A simplified HPLC method for tolbutamide metabolism to hydroxytolbutamide has been used to screen sixty psychoactive drugs for their ability to inhibit rat liver microsomal tolbutamide hydroxylation. One-step extraction with diethyl ether was followed by reconstitution and isocratic HPLC analysis with a binary mobile phase (ammonium phosphate:methanol, 45:55, v/v). Nanogram amounts of hydroxytolbutamide formation were estimated with UV detection at 240 nm. Hydroxytolbutamide formation was linear with incubation times of 40-120 min, but specific activity increased with increases in microsomal protein (0.15-1.10 mg). A differential inhibitory response was demonstrated for tolbutamide and debrisoquine hydroxylation to 5 psychoactive drugs, suggesting that tolbutamide hydroxylation is not dependent on P4502D1. Sixty psychoactive drugs, or drug metabolites, (at 33 microM) were then co-incubated with tolbutamide (at 2.5 and 10.2 microM). Tolbutamide hydroxylation was refractory (< 25% inhibition) to twenty-four of the drugs and only mildly inhibited (25-50% inhibition) by twenty-eight. Two compounds, trans-3-methylfentanyl and flurazepam, produced > 50% inhibition that was independent of tolbutamide concentration. Five of the drugs (methadone, chlorpheniramine, meperidine, 6-monoacetylmorphine and methylphenidate), however, caused greater than 50% inhibition in a competitive manner which suggests these drugs may share an affinity for the substrate binding site for tolbutamide.

Effect of phenobarbital treatment on carbon tetrachloride-mediated cytochrome P-450 loss and diene conjugate formation.

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.

Disturbances in hepatic heme metabolism in rats administered alkyl halides.

Liver tissue from rats administered 13 different alkyl halides and 4 other hepatotoxins were assayed for indices of hepatic heme synthesis. These included aminolevulinic acid (ALA)-dehydratase activity, porphyrin content, microsomal cytochrome P-450 and reduced glutathione content. Consistent decreases in ALA-dehydratase activity (14 of 17 compounds) and cytochrome P-450 content (14 of 17 compounds) were found. Significant changes in glutathione and porphyrin content also occurred after exposure to some compounds, but were not consistent. Alkyl halides are a class of toxicologically important compounds which have been found to exert an influence on hepatic heme synthesis.