Developmental toxicology studies of vancomycin hydrochloride administered intravenously to rats and rabbits.

Pregnant CD rats were given vancomycin intravenously in doses of 0, 40, 120, or 200 mg/kg on Gestation Days (GD) 6-15; pregnant New Zealand white rabbits were given 0, 40, 80, or 120 mg/kg intravenously on GD 6-18. Cesarean sections were performed on rats and rabbits on GD 20 and 28, respectively. In rats, maternal toxicity was indicated in the 120- and 200-mg/kg treatment groups by cortical tubular nephrosis. Maternal body weight gain and food consumption and fetal viability, weight, and morphology were not adversely affected by vancomycin. Maternal and developmental no observed adverse effect levels (NOAELs) in the rat were 40 and 200 mg/kg, respectively. In rabbits, maternal toxicity was indicated by cortical tubular nephrosis in the 80- and 120-mg/kg treatment groups; a single death and depression of body weight gain and food consumption occurred in the 120-mg/kg treatment group. Developmental toxicity was indicated by depression of fetal weight in the 120-mg/kg treatment group; fetal viability and morphology were not adversely affected by vancomycin. Maternal and developmental NOAELs in the rabbit were 40 and 80 mg/kg, respectively. Based on these data, vancomycin did not exhibit selective toxicity toward the developing rat or rabbit conceptus.

Cardiovascular and autonomic pharmacology of the macrolide antibiotic LY281389 in anesthetized beagles and in isolated smooth and cardiac muscles.

As part of the preclinical safety evaluation process, an investigational macrolide antibiotic, LY281389, was examined for autonomic activity in isolated smooth and cardiac muscle preparations and for cardiovascular effects by intravenous infusion in anesthetized beagles. Concentration-dependent antagonism of acetylcholine and angiotensin I (guinea pig ileum), norepinephrine (rat vas deferens), and isoproterenol (guinea pig atria) was observed at LY281389 concentrations of greater than or equal to 10(-5) M. At LY281389 concentrations of greater than or equal to 10(-4) M, the response of the guinea pig ileum to electrical stimulation was also inhibited approximately 65 to 100%, indicative of potential anticholinergic or alpha-adrenergic activity. In anesthetized dogs, the predominant effect of LY281389 was an increase in heart rate at doses of greater than or equal to 200 micrograms/kg of body weight. LY281389 also produced slight increases in mean arterial pressure and shortening of the P-R interval of the electrocardiogram. In summary, LY281389 possesses nonselective receptor antagonist activity in vitro and produces cardiovascular stimulation in anesthetized dogs. These results indicate that, in addition to potent antimicrobial activity, the macrolide antibiotic LY281389 may exert unexpected actions on cardiovascular function.

Comparative effects of disulfiram and N-methyltetrazolethiol on spermatogenic development in young CD rats.

N-Methyltetrazolethiol (NMTT) and NMTT-containing cephalosporin antibiotics cause characteristic testicular lesions in young but not adult rats. In addition, NMTT-containing cephalosporins inhibit aldehyde dehydrogenase and have been associated with a disulfiram-like reaction in humans and animals. Therefore, the potential testicular toxicity of disulfiram (10, 30, or 100 mg/kg) was evaluated in 37-day-old rats given oral doses on Postpartum Days 6 through 36, and was compared to the toxicity induced by NMTT (100 mg/kg). NMTT and each dose of disulfiram caused a decrease in testes weight. By DNA flow cytometry, testicular cell suspensions from rats given 100 mg/kg of NMTT had a 40% reduction in spermatids while those from rats given 10, 30, or 100 mg/kg of disulfiram had reductions of 52, 61, or 89%, respectively. Microscopically, the testes of rats given either NMTT or disulfiram had qualitatively similar changes, characterized by delayed maturity of the leading waves of germinal cells which had reached early maturation phase in control animals. Moderate to severe reduction occurred in the total number of spermatids with complete absence of acrosome phase and maturation phase spermatids. There was also a prominent reduction in the number of spermatocytes. Reduction in number of spermatogonia was minimal. While the mechanism of toxicity is not known for either compound, it is possible that the toxicity was related to the enzyme-inhibitory effects which both compounds possess. By defining the mechanism of testicular toxicity for compounds which cause a NMTT-like testicular toxicity in rats, biological differences in the spermatogenic process between the young and adult rat may be further understood. Direct extrapolation of the testicular effects in neonatal rats to man is not possible because of the substantial differences in initiation of spermatogenesis between rodents and humans.

Comparison of in vitro and in vivo models to assess venous irritation of parenteral antibiotics.

The venous irritation potential of four parenteral antibiotics, tetracycline hydrochloride (TET), erythromycin lactobionate (ERY), amphotericin B (AMP), and cephaloridine (CEP), was evaluated in an in vivo model using the rabbit ear vein. Lateral ear veins of New Zealand White rabbits were infused for 1 hr with test solutions containing TET (0.25,2.5, or 10 mg/ml), ERY (2.5, 5, or 25 mg/ml), AMP (0.05, 0.1, or 0.5 mg/ml), or CEP (4 or 20 mg/ml). Control rabbits received comparable volumes of 0.9% NaCl or 5% dextrose. Approximately 24 hr postinfusion, the rabbits were evaluated for visually evident changes in the treated ears. Pathologic evaluation of the veins was performed using histologic sections and scanning electron microscopy. TET, ERY, and AMP caused concentration-dependent changes in veins characterized primarily by loss of endothelium with associated inflammation and thrombus formation, consistent with the known clinical irritancy of these antibiotics. CEP, on the other hand, was well tolerated in the rabbit ear vein, paralleling its low irritancy potential in man. Test solutions identical to those used in vivo in rabbits were also evaluated in established in vitro assays for hemolytic potential when mixed with whole blood from monkeys and for damage to L6 muscle cells as determined by loss of creatine phosphokinase. Results of the in vitro test systems paralleled those of the rabbit ear model, with TET, ERY, and AMP exhibiting dose-dependent hemolysis and muscle cell toxicity, while CEP was comparatively nontoxic. Of the three models, the rabbit ear vein had the greatest sensitivity when histopathologic evaluation was employed.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative toxicity of oral cephalosporin antibiotics in the rabbit and in a rabbit kidney cell line (LLC-RK(1)).

The nephrotoxic potential of four oral cephalosporin antibiotics, cephalexin, cefaclor, LY195885 and LY171217, was determined in rabbits given single oral doses of 250-500 mg/kg body weight. Histopathological changes, blood chemistry, and ex vivo renal slice function were evaluated 48 hr after dosing. Additionally, the viability of rabbit renal cells in culture (LLC-RK(1)) was evaluated by nigrosin dye exclusion after 48 hr exposure to each antibiotic at concentrations of 0.5-2.0 mg/ml. Only LY171217 was significantly nephrotoxic in vivo. Prominent lesions were observed at 500 mg/kg body weight and were accompanied by marked increases in blood urea nitrogen and serum creatinine, and decreases in ex vivo renal slice gluconeogenesis and p-aminohippurate and tetraethylammonium uptake. In vitro toxicity to renal cells correlated well with the in vivo results yielding TC(50) values (TC(50) = concentration producing 50% lethality) > 1.0 mg/ml for cephalexin, LY195885 and cefaclor. LY171217, on the other hand, was significantly toxic in vitro (TC(50) = < 0.5). These results suggest that renal cells in culture may provide a useful method for examining the nephrotoxic potential of oral cephalosporins before in vivo studies.

Effects of cefamandole on spermatogenic development of young CD rats.

The testicular toxicity of cefamandole, a beta-lactam antibiotic with an N-methylthiotetrazole side chain, was evaluated in neonatal rats. Cefamandole caused delayed maturity of the germinal epithelium of neonatal rats when given on Postpartum Days 6 through 36. In rats given daily subcutaneous injections of 1000 mg/kg during this period, the most mature germinal cells were acrosome phase spermatids, whereas control rats had spermatids in the maturation phase. In studies of specific developmental phases, the effect of 1000 mg/kg daily cefamandole was primarily on the initial waves of spermatogonia during the period of rapid development (Postpartum Days 4 through 13). In animals treated from birth to Postpartum Day 8 and evaluated sequentially on Postpartum Days 5 through 9, there were no morphologically discernible effects on the transformation of gonocytes to immature spermatogonia, but there were slight degenerative changes in the first waves of developing spermatogonia. Cefamandole, 1000 mg/kg daily, given Postpartum Days 14 through 18 during the initial phase of spermatocyte development, also caused a slight degenerative change of the initial waves of pachytene spermatocytes. The significance of the findings in neonatal rats is unknown because differences in spermatogenic development between rat and human preclude direct extrapolation of the effects of cefamandole in neonatal rats to effects in humans.

Fluorine substitution as a probe for the role of the 6-position of benzo[a]pyrene in carcinogenesis.

The tumorigenic activities of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-F-BP) were compared to determine whether an unsubstituted 6-position is important for the carcinogenic effect of BP. Highly purified samples of 6-F-BP and BP had similar activities for the induction of lung adenomas in Swiss Webster mice treated before weaning. The 6-fluoro derivative, however, had about one-half as much activity as BP for the initiation of skin papillomas in CD-1 mice. Similarly, 6-F-BP (approximately equal to 90% purity) had about one-half the activity of BP for the induction of skin tumors in C57BL/6J mice given repetitive treatments of the hydrocarbons and for the induction of sarcomas in C3H/fCum mice given a single sc injection. 6-F-BP (approximately equal to 90% purity) had activity similar to that of BP for induction of sarcomas at the sc injection site in Fischer 344 rats. These results and related data indicate the need for detailed metabolic studies whenever fluorine substitution is used as a probe to assess the role of the unsubstituted position in the carcinogenicity of the parent compound.

Ethanol-moxalactam interactions in vivo.

Adverse reactions similar to disulfiram reactions observed in volunteers given moxalactam prompted an investigation to determine whether moxalactam, like disulfiram, leads to an accumulation of acetaldehyde during ethanol metabolism. Concentrations of ethanol and acetaldehyde in blood of male Wistar rats given test compounds and ethanol were determined by gas chromatography. Moxalactam, like disulfiram, had no effect on concentrations of ethanol but increased the concentrations of acetaldehyde. However, the effect after treatment with moxalactam was less than after treatment with disulfiram. The interval between pretreatment with moxalactam and administration of ethanol that gave the maximal effect ranged from 3 to 24 hr. When ethanol was given before or at the same time as moxalactam, no effect was observed. Cefamandole and cefoperazone, which, like moxalactam, have a methyltetrazolethiol side chain, increased concentrations of acetaldehyde, but penicillin G, carbenicillin, cephalothin, cephradine, cefoxitin, cefazolin, and cefotaxime had no effect. For prevention of a disulfiram-like reaction, physicians should caution patients to avoid alcoholic beverages for several days after treatment with moxalactam, cefamandole, or cefoperazone.

Activation and inhibition of benzo(a)pyrene and aflatoxin B1 metabolism in human liver microsomes by naturally occurring flavonoids.

The effects of several naturally occurring and synthetic flavonoids on the metabolism of benzo(a)pyrene and aflatoxin B1 were evaluated. Addition of apigenin, chrysin, fisetin, flavonone, galangin, hesperitin, kaempferol, morin, myricetin, haringenin, or quercetin to human liver microsomes inhibited the hydroxylation of benzo(a)pyrene. In contrast to these results, the addition of flavone, nobiletin, tangeretin, or 7,8-benzoflavone to human liver microsomes caused a many-fold stimulation in the hydroxylation of benzo(a)pyrene, the metabolism of aflatoxin B1 to 2,3-dihydro-2,3-dihydroxyaflatoxin B1, and the metabolic activation of aflatoxin B1 to mutagenic products. Quercetin, morin, and kaempferol inhibited cytochrome c (P-450) reductase in human liver microsomes whereas flavone and 7,8-benzoflavone had no effect. These results suggest that the inhibitory effects of quercetin, morin, and kaempferol on monooxygenase activity may be caused at least in part by an inhibition in the reduction of cytochrome P-450. An examination of the structural features required for the inhibition and stimulation of benzo(a)pyrene hydroxylation indicated that all of the 12 flavonoid inhibitors that were studied possessed hydroxyl groups whereas the flavonoid activators were less polar molecules that lacked hydroxyl groups.

An enantiomeric interaction in the metabolism and tumorigenicity of (+)- and (-)-benzo[a]pyrene 7,8-oxide.

The (+)- and (-)-enantiomers of benzo[a]pyrene 7,8-oxide are hydrated stereospecifically at C-8 to (-)- and (+)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, respectively, by rat hepatic epoxide hydrolase. The (-)-enantiomer of benzo[a]pyrene 7,8-oxide is metabolized by microsomal epoxide hydrolase at a rate 3- to 4-fold greater than the (+)-enantiomer. At low conversion of racemic substrate, however, benzo[a]pyrene 7,8-oxide is metabolized to the dihydrodiol at a rate equal to that of the (+)-enantiomer. An analysis of the enantiomeric composition of the dihydrodiol formed from the racemic substrate revealed preferential formation of (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo-[a]pyrene. At low substrate conversion (< 20% metabolism), the enantiomeric purity of the dihydrodiol was much higher than at high substrate conversion (> 50% metabolism). Similar results were obtained with microsomes from hamster, rabbit, guinea pig, mouse, and human liver. These results indicate that epoxide hydrolase has a higher affinity for (+)-benzo[a]pyrene 7,8-oxide than for the (-)-enantiomer. The kinetics of hydration of (+)- and (-)-benzo[a]pyrene 7,8-oxide by purified epoxide hydrolase in detergent solution showed the (+)- and (-)-enantiomers to have apparent Km values of 1.7 and greater than or equal to 20 microM, respectively. Tumorigenicity studies with benzo[a]pyrene 7,8-oxide on mouse skin and in newborn mice revealed that (+)-benzo[a]pyrene 7,8-oxide, the metabolic precursor of the more tumorigenic (-)-7,8-dihydrodiol, is significantly more tumorigenic than the (-)-enantiomer. However, racemic benzo[a]pyrene 7,8-oxide was more tumorigenic than either enantiomer alone, indicating an enantiomeric synergism in the carcinogenicity of benzo[a]pyrene 7,8-oxide. The data are discussed in relation to the complete sequence of metabolic pathways leading to an ultimate carcinogen from benzo[a]pyrene.

Tumorigenic activity of benzo(e)pyrene derivatives on mouse skin and in newborn mice.

The tumorigenic activities of benzo(e)pyrene and several of its derivatives were determined in two mouse tumor models. Newborn Swiss-Webster mice were given i.p. injections of 0.4, 0.8, and 1.6 mumol of compound on the first, eighth, and 15th day of life, respectively. When the mice were 62 to 66 weeks old, the experiment was terminated by killing the animals. Benzo(e)pyrene, trans-4,5-dihydroxy-4,5-dihydrobenzo(e)pyrene, and trans-9,10-dihydroxy-9,10-dihydrobenzo(e)pyrene had little or no tumorigenic activity in lung tissue, although trans-9,10-dihydroxy-9,10-dihydrobenzo(e) pyrene did induce a significant number of hepatic tumors. The tumor-initiating activities of benzo(e)pyrene and several of its derivatives were determined on the skin of female CD-1 mice. A single topical application of 1.0 to 6.0 mumol of the test compound was followed 7 days later by twice-weekly applications of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate for 35 weeks. Control mice and mice treated with 6.0 mumol of benzo(e)pyrene, trans-4,5-dihydroxy-4,5-dihydrobenzo(e)pyrene, trans 9,10-dihydroxy-9,10-dihydrobenzo(e)pyrene, and trans-9,10-dihydroxy-9,10,11,12-tetrahydrobenzo(e)pyrene had a tumor incidence of less than 20% and had less than or equal to 0.25 papillomas/mouse. 9,10-Dihydrobenzo(e)pyrene was the only derivative tested that had significant tumor-initiating activity on mouse skin; an initiating dose of 2.5 mumol gave a 67% tumor incidence and 1.43 papillomas/mouse.

Regulation of human drug metabolism by dietary factors.

Several dietary factors influence the oxidative metabolism of chemicals in humans. Increasing the ratio of protein to carbohydrate or fat in the diet, feeding cabbage and brussels sprouts or feeding charcoal-broiled beef for several days stimulates human drug metabolism. The chronic ingestion of ethanol stimulates drug metabolism whereas the chronic ingestion of methylxanthine-containing foods inhibits drug metabolism. In contrast, an increase in the ratio of fat to carbohydrate in the diet of normal subjects or the fasting of obese individuals for several days has little or no effect on drug metabolism. Flavonoids in edible plants influence the metabolism of foreign chemicals by human liver in vitro. The addition of flavone, tangeretin or nobiletin to human liver microsomes activates both the hydroxylation of benzo[alpha]pyrene and the metabolism of aflatoxin B1 to mutagens. On the other hand, quercetin, kaempferol, morin and chrysin, which are also normally occurring flavonoids, inhibit the hydroxylation of benzo[alpha]pyrene by human liver microsomes.

Tumorigenicity of the diastereomeric benz[a]anthracene 3,4-diol-1,2-epoxides and the (+)- and (-)-enantiomers of benz[a]anthracene 3,4-dihydrodiol in newborn mice.

The tumorigenic activity of benz[a]anthracene (BA), the (+)- and (-)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BA 3,4-dihydrodiol), and the racemic diastereomers of the BA 3,4-diol-1,2-epoxides [i.e., either or both of the diastereomeric 1,2-epoxides derived from BA 3,4-dihydrodiol in which the epoxide oxygen is cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 4-hydroxyl group) was examined in newborn Swiss-Webster mice. The mice were administered ip a total dose of 280 nmoles of compound in divided doses consisting of 40 nmoles within 24 hours of birth, 80 nmoles at 8 days of age, and 160 nmoles at 15 days of age. The experiment was terminated when the animals were 26 weeks of age. BA 3,4-diol-1,2-epoxide-2 was the most potent compound tested. All animals treated with BA 3,4-diol-1,2-epoxide-2 developed pulmonary tumors with an average of 13.3 tumors per mouse. BA 3,4-diol-1,2-epoxide-1 produced pulmonary tumors in 42% of the mice with an average of only 0.56 tumors per mouse. The (-)-enantiomer of BA 3,4-dihydrodiol with [3R,4R] absolute stereochemistry was the second most tumorigenic derivative of BA tested; it produced pulmonary tumors in 71% of the mice with an average of 1.88 tumors per mouse. BA and the (+)-enantiomer of BA 3,4-dihydrodiol had little or no tumorigenic activity at the dose tested. A comparison of the average number of pulmonary tumors per mouse revealed that BA 3,4-diol-1,2-epoxide-2 was about 30-fold more tumorigenic than was BA 3,4-diol-1,2-epoxide-1, 8-fold more tumorigenic than was (-)-BA 3,4-dihydrodiol, and greater than 85-fold more tumorigenic than was BA. These data indicate that in newborn mice BA 3,4-dihydrodiol and a BA 3,4-diol-1,2-epoxide are proximate and ultimate carcinogenic metabolites of BA, respectively.

Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The tumorigenicities of benzo[a]pyrene and each optical enantiomer of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides derived from trans-7,8-dihydroxy-7,8-dihydrobenzol[a]pyrene were tested by sequential intraperitoneal injection of mice with 1,2, and 4 nmol, or with 2, 4, and 8 nmol of each compound on the 1st, 8th, and 15th day of life, respectively. The experiment was terminated when the animals were 34--37 weeks old. (+)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2] had exceptional tumorigenicity, whereas benzo[a]-pyrene and the other three optically pure isomers of the benzo[a]pyrene 7,8-diol,9,10-epoxides had little or no activity. These results demonstrate differences in the carcinogenic activities of optically active isomers of a polycyclic hydrocarbon diol epoxide. Eleven percent of control mice had pulmonary tumors, whereas 71% and 100% of the mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2, respectively, had pulmonary tumors. Control mice had an average of 0.12 pulmonary tumors per mouse, whereas mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2 had 1.72 and 7.67 pulmonary tumors per mouse, respectively. Mice treated with 14 nmol of (-)-BP-7alpha,8beta-diol-9beta,10beta-epoxide 2, (-)-BP-7beta,8alpha-diol-9beta,10beta-epoxide 1, or (+)-BP-7alpha,8beta-diol-9alpha,10alpha-epoxide 1 had 0.13, 0.25, and 0.34 pulmonary tumors per animal, respectively.