The relationship among microsomal enzyme induction, liver weight and histological change in beagle dog toxicology studies.

The present study represents a retrospective analysis of hepatic microsomal enzyme induction data collected over a period of years for the beagle dog. Comparisons were completed for up to six enzyme activities and P450 content versus histopathological examination of the liver for hepatic changes and serum chemistry data analysis for markers indicative of hepatic injury. In addition, qualitative comparisons were made for these compounds to data reported in the rat by the same authors. In this analysis of canine study data for nine different compounds comprising five different pharmacological classes, significant elevations in several microsomal enzyme activities were observed under study conditions that did not result in liver weight increases, histological changes or serum chemistry changes that would be indicative of hepatocellular or hepatobiliary damage. Despite some species differences in cytochrome P450 homologues, for this compound set, there was clearly a general association between the response in dog liver and that of the rat liver. Compounds that elicited significant increases in more than one canine P450 endpoints were also likely to produce an inductive response in rat liver; however, the magnitude of the response and the P450 endpoint involved were not always identical. We conclude that hepatic drug-metabolizing enzyme induction in the beagle dog liver is typically a benign adaptive response, which parallels that reported previously in the rat.

A comparison of the direct and reporter antigen popliteal lymph node assay for the detection of immunomodulation by low molecular weight compounds.

The direct popliteal lymph node assay (PLNA) is a predictive test used to detect the immune-stimulating potential of pharmaceuticals and other low molecular weight compounds (LMWCs) with known autoimmunogenic or sensitizing properties. Two limitations in the PLNA are the existence of false negatives and the inability of the assay to provide mechanistic information. Recently the direct PLNA was modified by incorporating reporter antigens (RA), either TNP-Ficoll or TNP-OVA. In the RA-PLNA, immune stimulation is detected by measuring IgM or IgG TNP-specific antibody-forming cells (AFC) using an enzyme-linked immunospot (ELISPOT) assay. The RA-PLNA, when using potent, known autoimmunogenic compounds, may provide greater sensitivity compared to the direct PLNA and might distinguish LMWCs that have intrinsic adjuvant activity from those that create neo-antigens, using TNP-OVA and TNP-Ficoll, respectively. The purpose of this study was to rigorously compare the two assays. Our first objective was to investigate the interlaboratory reproducibility of the RA-PLNA using four autoimmunogenic LMWC models, plus one negative control LMWC. Subsequently, we tested seven LMWCs with known sensitizing properties and compared the results from the direct and modified assay. The test group included LMWCs thought to be mechanistically distinct and similar to compounds typically encountered in preclinical safety assessment. All control and treatment AFC plaques were collected (76 total), pooled, coded to conceal their source, and counted. The interlaboratory reproducibility of the RA-PLNA was demonstrated with the model autoimmunogenic compounds HgCl2, diphenylhydantoin, D-penicillamine, and the negative control compound phenobarbital, by detecting TNP-specific IgM and polyclonal IgG production to both reporter antigens. Additionally, the sensitizing effects of streptozotocin were identified using an IgG2a ELISPOT with both TNP-OVA and TNP-Ficoll. With the extended test group, the sensitizing effects of aniline, a false negative LMWC in the direct PLNA, was not detected in this study when using the direct PLNA. However, there was an increase of IgG1 AFCs using TNP-OVA, when compared to control (508 +/- 113 vs. 12 +/- 4 respectively). Glafenine, diclofenac, and ibuprofen, all associated with drug-induced anaphylaxis in humans, produced significant increases in IgG1 production to TNP-OVA. Of these three LMWCs, only diclofenac, which has been documented to induce neo-antigen formation, was detected with TNP-Ficoll. Hydralazine immunomodulation could be detected only with the direct PLNA although significant increases in IgM were identified with the co-injection of either reporter antigen. Isoniazid and methyldopa consistently produced negative responses in both assays. In summary, this study has demonstrated acceptable interlaboratory reproducibility of the RA-PLNA, using model autoimmunogenic LMWCs. Additionally, it demonstrated that an advantage of the RA-PLNA was that it identified all anaphylactic-associated LMWCs tested, detected the false negative compound aniline, and revealed what is thought to be the mechanism(s) associated with diclofenac-induced immunostimulation.

The relationship among microsomal enzyme induction, liver weight and histological change in rat toxicology studies.

The purpose of this study was to determine what histological changes, if any, accompany liver enlargement and microsomal enzyme induction in rats administered high doses of therapeutic agents in preclinical toxicology studies. This was accomplished by evaluating a database derived from a series of 11 induction studies in rats with 10 novel compounds comprising five therapeutic classes. Results from serum enzyme chemistry analyses, gross organ weight changes, and histological analyses of the liver sections were evaluated and compared with the magnitude and extent of hepatic cytochrome P450 induction. All compounds were administrated via oral intubation once a day for the duration of the study using multiple doses, each proportionally based on body weight. During the course of these studies, serum clinical chemistry data and clinical observations were recorded. After necropsy, histopathology observations were made, and hepatic microsomes were assayed for cytochrome P450 content and associated drug-metabolizing enzymes. In some cases, cyanide-insensitive beta-oxidation of palmitoyl CoA was also assayed. Liver weight increases of 20% or greater were associated with histological evidence of hypertrophy, but neither the severity of hypertrophy nor the magnitude of liver weight increase correlated with the magnitude of drug-metabolizing enzyme elevations. Hypertrophy alone was not associated with serum enzyme increases. While there was a correlation between the incidence of increased liver weights and microsomal enzyme induction, the magnitudes of these increases were not related. Decreased serum triglycerides were often associated with elevated beta-oxidation attributed to hepatic peroxisome proliferation. It was concluded that, while slight ALT elevations occasionally were observed, hepatic microsomal enzyme induction was generally not accompanied by substantial morphological changes or elevated serum enzyme levels considered indicative of liver injury.

Ethylmorphine N-demethylase activity as a marker for cytochrome P450 CYP3A activity in rat hepatic microsomes.

The purpose of this study was to evaluate the selectivity and sensitivity of ethylmorphine N-demethylase (EMD) as an indicator of chemically-induced cytochrome P450 CYP3A activity in liver microsomes of rats following treatment with selective enzyme inducers. Male and female Sprague-Dawley (CD) rats were dosed with either pregnenolone-16alpha-carbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone (betaNF; 100 mg/kg per day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), isoniazid (ISO; 100 mg/kg per day for 3 days), or dexamethasone (DEX; 50 mg/kg per day for 4 days). Microsomes were isolated, frozen and subsequently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations (P < 0.01) in EMD activity were observed in microsomes from PB-, DEX- and PCN-dosed animals compared with untreated controls. Microsomes from ISO- and betaNF-dosed males showed a reduction (P < 0.05) in EMD activity when compared with control microsomes, and CF was without effect. In females, EMD activities were significantly increased in microsomes from PCN, DEX and PB-dosed but not betaNF, ISO, or CF-dosed animals. As expected on the basis of sex-related differences in gene expression, EMD activities in untreated animals were considerably higher in males than females, attributable to constitutive CYP3A and CYP2C11 activities. The selectivity of EMD for induced CYP3A was confirmed on the basis of inhibition studies with selected steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclusion, for both sexes, the greatest elevations (approximately 3-13-fold) in EMD activity were observed in microsomes from rats dosed with DEX, a potent archetypal inducer with lesser but significant increases noted for PCN and PB, indicating that EMD is a reliable indicator of induced rat hepatic cytochrome P450 CYP3A activity.

Hepatic microsomal enzyme induction, beta-oxidation, and cell proliferation following administration of clofibrate, gemfibrozil, or bezafibrate in the CD rat.

Male and female CD rats were administered one of two dose levels of clofibrate, gemfibrozil, or bezafibrate daily by oral gavage for a period of 14 days in order to establish an empirical data base using the Charles River CD rat with a single class of drugs against which the potency of novel proprietary compounds could be compared. Subsequent gross examination of the liver indicated significant and dose-related increases in relative and absolute liver weights in males following clofibrate and gemfibrozil. In females, absolute and relative liver weights were significantly elevated to a similar degree with either dose of gemfibrozil, and absolute liver weights were higher in clofibrate-dosed animals. Bezafibrate had no effect on female liver weights. Clofibrate and gemfibrozil increased hepatic palmitoyl CoA beta-oxidation in both sexes; however, clofibrate had the greater effect in males and gemfibrozil had the least effect in females. Bezafibrate treatment resulted in a very pronounced elevation of palmitoyl CoA beta-oxidation in the males but had no similar effect in the females. Concurrent ELISA analysis for cytochrome CYP4A revealed very good correspondence between beta-oxidation and cytochrome induction for each of the three compounds in males, but other cytochromes were not greatly affected, except CYP1A1 which was elevated in bezafibrate-dosed females. For males, further analysis for markers of cellular proliferation, namely cyclin-dependent kinases (CDK) and proliferating cell nuclear antigen (PCNA), indicated dose-related increases for both with clofibrate, increases at the high dose for gemfibrozil, and, for PCNA, a dose-related increase for bezafibrate. In females, both markers for cell proliferation showed either slight or no increases following any of the three drug treatments. These results demonstrate clear sex-dependent differences in terms of relative potency in the hepatic response of the Sprague-Dawley-derived rat to these peroxisome proliferators. Bezafibrate is most potent and gemfibrozil is least potent in stimulating peroxisome-associated beta-oxidation and cytochrome P450 4A induction in the males. Even though gemfibrozil significantly increased liver weights, beta-oxidation and cytochrome P450 4A in the females increased only after clofibrate treatment, although to a lesser degree than in the males administered the same dose. Similar sex-related differences were observed for cell proliferation. In conclusion, sex-related differences were noted in the potency to stimulate acyl Co-A oxidation, its association with hepatomegaly, and the stimulation of cell proliferation, but CYP4A induction always accompanied any substantial drug-dependent increases in beta-oxidation.

Effects of hexachlorophene on myelin marker enzymes in rat oligodendrocytes.

The antibacterial substance hexachlorophene (HCP) can affect myelin formation or integrity leading to intramyelinic oedema and vacuolation in the central nervous system through an unknown mechanism. These studies were conducted to investigate the direct, dose-dependent effects of HCP on myelin membrane markers in cultured oligodendrocytes (OLG) isolated from 4-7-day-old rat pups and cultured in vitro for up to 5 wk. 2-wk-old OLG cultures were exposed to 0, 0.24 or 0.74 mum HCP for 48 hr. At 48 hr and again at 5, 12 and 19 days after the end of dosing the myelin markers galactosylceramide (GalC), myelin basic protein (MBP), and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) were quantified by ELISA or biochemical techniques. DNA was measured to estimate total cell mass and astrocyte contamination was determined by an ELISA procedure using anti-glial fibrillary acidic protein (GFAP) as the primary antibody. Because of the use of a selective culture medium, astrocyte contamination was initially low and continued to decrease from wk 2 to 4 as determined by GFAP binding. CNPase, GalC and MBP levels were similar in control and low-dose (0.24 mum HCP) cultures with a general increase in MBP and CNPase over time. Cultures exposed to 0.74 mum HCP showed a decline in GalC proportional to decreased DNA content with time, but levels of MBP and CNPase increased after dosing and were always greater than the corresponding levels in control or low-dose cultures. These studies suggest a direct, dose-related toxic effect of HCP accompanied by a stimulation of MBP and CNPase but not of GalC production in the membranes of the recovering OLG following removal of HCP.

Comparison of the effects of the new azalide antibiotic, azithromycin, and erythromycin estolate on rat liver cytochrome P-450.

Erythromycin and some other macrolide antibiotics can first induce a cytochrome P-450 isozyme similar to the one induced in rats by pregnenolone-16 alpha-carbonitrile and then inhibit it by forming a stable cytochrome P-450-metabolite complex. The purpose of this study was to compare azithromycin, a novel 15-membered ring azalide, and erythromycin estolate for the potential to cause hepatic microsomal enzyme induction and inhibition in Sprague-Dawley rats. The daily oral administration of 800 mg of erythromycin estolate per kg for 7 days resulted in statistically significant elevations of NADPH-cytochrome c reductase, erythromycin N-demethylase (3.2-fold), and total cytochrome P-450 content. Approximately 40% of cytochrome P-450 was complexed with erythromycin metabolite. In contrast, the daily administration of 200 mg of azithromycin per kg for 7 days caused significant elevations of N-demethylase (2.5-fold) only and did not produce any increases in total cytochrome P-450 content or NADPH-cytochrome c reductase. No complexed cytochrome P-450 was detected in the azithromycin-dosed rats despite liver concentrations of azithromycin that were 118 times greater than the liver concentrations of erythromycin estolate in erythromycin estolate-dosed rats. Although the short-term oral administration of azithromycin produced hepatic accumulation of the drug and elevated azithromycin demethylase activity, there was no other evidence of hepatic cytochrome P-450 induction or inactivation via cytochrome-metabolite complex formation. In contrast to erythromycin estolate, azithromycin is not expected to inhibit its own metabolism or that of other drugs via this pathway.

Selective membrane toxicity of aminoglycoside antibiotics in membrane vesicles isolated from proximal renal tubules of the rat.

A considerable body of evidence suggests that the nephrotoxic potential of aminoglycoside antibiotics may be associated with the degree of membrane binding and subsequent membrane damage in the renal tubules. In this study, we isolated functional basolateral and luminal membrane vesicles from rat renal cortex, incubated each membrane type in the presence of 1 mM concentrations of either neomycin, netilmicin, gentamicin, hydroxygentamicin, or amikacin, and monitored the activities of the marker enzymes alkaline phosphatase (ALP) and lambda-glutamyltransferase (GGT) (luminal) or ouabain-sensitive Na+,K+-ATPase (basolateral) to determine if there were any selective drug-related alterations of enzyme activities. While none of the five aminoglycosides had any substantive effect upon enzyme activities of luminal vesicles, all five drugs inhibited the basolateral marker enzyme. Neomycin produced the greatest inhibition, hydroxygentamicin and amikacin the least, and gentamicin and netilmicin were intermediate in the inhibition of the enzyme. These results are in accordance with the known relative nephrotoxicity of these same drugs and indicate the usefulness of isolated renal membrane vesicles for in vitro toxicological studies of novel aminoglycosides.

Further characterization of serum alkaline phosphatase from male and female beagle dogs.

Alkaline phosphatase (AP) from the sera of both male and female beagle dogs was partially purified and then analyzed for the presence of AP isoenzymes having intestinal or osseous characteristics as detected by bromotetramisole inhibition or wheat germ lectin agarose electrophoresis, respectively. The sera from both sexes were similar in regard to the presence of AP isoenzymes with intestinal (16 vs. 20%) or osseous (19 vs. 23%) characteristics, but serum AP from the male had a greater sialic acid content and only the male serum contained a detectable constitutive acidic (pI = 3.4) AP isoenzyme. This was similar to a serum AP isoenzyme previously found elevated in the sera of dogs afflicted with hyperadrenocorticalism or of dogs treated with certain corticosteroids.