Male and female B6C3F1 mice were dosed orally with AZT alone (100, 200, or 400 mg AZT/kg body weight per day), isoniazid alone (50, 100, or 150 mg/kg per day), or combinations of AZT and isoniazid for up to 94 days. Mice were evaluated for clinical signs, mortality, body and organ weights, sperm function and vaginal cytology, and hematology and clinical chemistry parameters. Core study mice, early-death clinical pathology mice, and females from the 400 + 150 mg/kg (AZT + isoniazid) clinical pathology group were necropsied and subjected to histopathological evaluations.The significant effects of treatment with AZT and isoniazid are summarized in the Abstract Table. The primary toxicity of AZT was bone marrow suppression. The hematopoietic toxicity was manifested by dose-related anemia, thrombocytosis, and a reticulocytopenia followed by reticulocytosis. Cellular depletion of the bone marrow was observed microscopically and was considered the major treatment-related effect. Increased pigmentation of the skin and a slight decline in epididymal sperm motility also occurred in mice treated with AZT alone.Administration of isoniazid alone resulted in slight hepatotoxicity manifested by hepatocellular hypertrophy and pigment deposition in the liver of male mice. Treatment with isoniazid also resulted in a slight increase in the duration of estrus.Administration of AZT in combination with isoniazid resulted in hematopoietic toxicity of greater severity than that resulting from the administration of AZT alone. The bone marrow suppression and anemia resulted in significant mortality in female mice treated with the highest combinations of AZT and isoniazid. Combination therapy also resulted in treatment-related declines in body weight, reticulocytopenia followed by reticulocytosis, thrombocytosis, leukopenia, neutropenia, lymphopenia, and a slight increase in the duration of estrus.[Table: see text]

The toxicity of combinations of AZT (200 or 400 mg) and pyrazinamide (1,000 or 1,500 mg) was evaluated in B6C3F1 mice treated by gavage for up to 94 days. The primary toxic effect of AZT was bone marrow suppression manifested by macrocytic anemia, thrombocytosis, and reticulocytopenia followed by reticulocytosis. Cellular depletion of bone marrow was observed microscopically. Administration of pyrazinamide alone caused mild hepatotoxicity, as evidenced by increased liver weights and by glycogen depletion of hepatocytes in a zonal pattern. AZT and pyrazinamide administered together resulted in a significant exacerbation of the hematopoietic toxicity induced by AZT alone. The hepatotoxicity of pyrazinamide was slightly augmented by AZT.

Combination therapy with anti-HIV drugs and opportunistic infection drugs is a common practice in treatment of AIDS patients. Although toxic effects of most individual therapies are known, the toxic potential of most combination therapies has not been established. To understand the toxic consequences of combination therapies, the commonly used anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) and tuberculosis infection therapies pyrazinamide, isoniazid, and rifampicin were evaluated by 13-week gavage studies in B6C3F1 mice, either alone or AZT in combination with one of the antituberculosis drugs. The doses include AZT 100, 200, and 400; pyrazinamide 1000 and 1500; isoniazid 50, 100, and 150; and rifampicin 100, 200, and 400 mg/kg/day, AZT alone caused hematopoietic toxicity with dose-related bone marrow suppression, macrocytic anemia, and thrombocytosis. Pyrazinamide or isoniazid alone at the doses tested did not cause significant toxicity. Rifampicin alone caused hematopoietic toxicity and possibly mild hepatic toxicity. Pyrazinamide below 10 times the therapeutic dose when given with AZT did not increase the hematological toxicity of AZT, Isoniazid markedly increased the hematological toxicity of AZT and contributed to mortality at 3 to 4 times the therapeutic dose combinations. Administration of rifampicin with AZT at the calculated therapeutic doses resulted in toxicity of far greater magnitude than that caused by AZT or rifampicin alone. Combination treatment with AZT and rifampicin caused severe anemia with mortality at 2 to 4 times the therapeutic dose combinations. However, AZT did not enhance the hepatotoxicity of rifampicin. Increased hematopoietic toxicity of AZT when given in combination with the above antituberculosis drugs may be due to changes in the metabolism of AZT, Results of these studies indicate that toxicological effects of combination therapies could be considerably more severe than and different from the toxicity of individual therapies,

Pyrazinamide is a synthetic pyrazine analogue of nicotinamide used in the treatment of tuberculosis, which is an opportunistic infection in the human immunodeficiency virus (HIV)-positive population. The reproductive and developmental toxicities of pyrazinamide were evaluated in male and female Swiss (CD-1®) mice by administering daily doses of 0, 400, 800, or 1,200 mg/kg of pyrazinamide in 0.5 % methyl cellulose in deionized water by gavage. Male mice (10 per group) were dosed on days 5 to 25 and sacrificed on day 25. Females were divided into two groups designated females-A and females-B. The females-A (20 per group) were dosed from day 0 to sacrifice and caesarean-sectioned on days 28 to 32 and were cohabited with dosed males on days 9 to 13 to test for effects on mating behavior, fertilization, and implantation. The females designated as females-B (20 per group) were cohabited with males on days 0 to 4, before the males began receiving pyrazinamide. Sperm-negative females-B were sacrificed after the cohabitation period; sperm-positive females-B were dosed during organogenesis on days 6 through 15 of presumed gestation. For females-B that delivered, both dams and pups were sacrificed on day 4 or 5 of lactation. Sperm-positive females-B that did not deliver pups were sacrificed on days 24 to 27 of presumed gestation. Adult mice were evaluated for clinical signs, body weights, pathologic findings at necropsy, and clinical pathology parameters. Sperm function was evaluated in males. From delivery until postnatal day 4, offspring from females-B were evaluated for viability, external anomalies, and body weight.No apparent clinical, reproductive, or developmental toxicity was detected at the doses employed in this study. A reduction occurred in female fetal body weight in females-A in the 800 and 1,200 mg/kg groups. Since this reduction was not highly significant (P≤0.05) in the 1,200 mg/kg group, it is uncertain whether this finding is related to treatment with pyrazinamide. The highest dose administered in female Swiss (CD-1®) mice, 1,200 mg/kg per day, is approximately 8 times the therapeutic dose and resulted in a Cmax 9 to 12 times the Cmax caused by the therapeutic dose in humans. However, results of this study indicated that higher doses could have been tolerated.

Groups of 10 male and 20 female B6C3F1 mice received 0, 500, or 1000 mg/kg/day 2'3'-dideoxycytidine (ddC) by gavage for 13 weeks. At the end of the 13-week exposure period all males and 10 females per group were necropsied while the remaining females were held for 1 month without further treatment. Thymic atrophy was present at the 13-week necropsy in male and female mice administered 1000 mg/kg/day and in females administered 500 mg/kg/day, but was not present in females following 1 month of recovery. Thymic lymphoma was present in 1 female that received 500 mg/kg/day and 1 female that received 1000 mg/kg/day. In a follow-up study groups of 70 female mice received 0, 500, or 1000 mg/kg/day for 13 weeks. At the end of the 13-week exposure period 20 mice per group were necropsied and the remaining animals held for 3 months without further treatment. Thymic atrophy was observed in ddC-exposed groups at the 13-week necropsy but not in mice allowed to recover for 13 weeks. Thymic lymphoma occurred in 3/50 mice that received 500 mg/kg/day and in 17/50 mice that received 1000 mg/kg/day but did not occur in mice from the vehicle control group.

t-Butyl alcohol (TBA) was administered in drinking water to F344/N rats and B6C3F1 mice for two years using 60 animals/dose/sex/species. Male rats received doses of 0, 1.25, 2.5, or 5 mg/ml and females received 0, 2.5, 5, or 10 mg/ml, resulting in average daily doses of approximately 85, 195, or 420 mg TBA/kg body weight for males and 175, 330, or 650 mg/kg for females. Ten rats per group were evaluated after 15 months. Male and female mice received doses of 0, 5, 10, or 20 mg/ml, resulting in average daily doses of approximately 535, 1,035, or 2,065 mg TBA/kg body weight for males and 510, 1,015, or 2,105 mg/kg for females. Survival was significantly reduced in male rats receiving 5 mg/ml, female rats receiving 10 mg/ml, and male mice receiving 20 mg/ml. Long-term exposure to TBA produced increased incidences of renal tubule adenoma and carcinoma in male rats; transitional epithelial hyperplasia of the kidney in male and female rats; follicular cell adenoma of the thyroid in female mice; and follicular cell hyperplasia of the thyroid and inflammation and hyperplasia of the urinary bladder in male and female mice. In addition, a slight increase in follicular cell adenoma or carcinoma of the thyroid (combined) in male mice may have been related to the administration of TBA.

The purpose of this study was to evaluate the toxicity of t-butyl alcohol, an important commodity chemical, an additive to unleaded gasoline, and a contaminant of drinking water. Ninety-day toxicity studies were conducted in B6C3F1 mice and Fischer 344 (F344) rats of both sexes using dosed water. Dose levels of t-butyl alcohol were 0, 0.25, 0.5, 1, 2, and 4% (w/v). Lethality was observed at the 4% level of both sexes and species. Weight-gain depression was present in all dose levels of male rats; 4% female rats; 1, 2, and 4% male mice; and 2 and 4% female mice. Water consumption was increased at lower dose levels in male rats and decreased in the higher dose levels of both sexes of rats and female mice. Clinical signs in rats were ataxia in both sexes and hypoactivity in males. Clinical signs in mice were ataxia, abnormal posture, and hypoactivity. In rats, urine volumes were reduced, in association with crystalluria. Gross lesions at necropsy were urinary tract calculi, renal pelvic and ureteral dilatation, and thickening of the urinary bladder mucosa. Microscopic lesions were hyperplasia of transitional epithelia and inflammation of the urinary bladder. In male rats treated with t-butyl alcohol, microscopic renal changes were suggestive of alpha-2 mu-globulin nephropathy. No-effect levels for the urinary tract lesions were 1% in male rats and mice (803.7 mg/kg/day for the male rats and 1565.8 mg/kg/day for the male mice) and 2% in female rats and mice (1451.5 mg/kg/day for the female rats and 4362.9 mg/kg/day for the female mice). The results indicate that in rodents the urinary tract is the target organ for t-butyl alcohol toxicity, and males are more sensitive to t-butyl alcohol toxicity than females.

Studies were conducted to define primary pharmacological and toxicological properties of two arotinoids, SMR-2 and SMR-6, in male B6D2F1 mice. Mice were gavaged daily for up to 22 days with retinoids in corn oil (0.1, 0.2, or 0.4 mg/kg day SMR-2 or SMR-6 or 2.5, 10, or 30 mg/kg all-trans-retinoic acid as a reference control). Toxicological and biochemical endpoints were assayed after 8, 15, and 22 days. At toxic doses, i.e., those inducing weight loss, morphological changes were observed in skin, lymph nodes, spleen, bone marrow, liver, thymus, forestomach, adrenal, bone, and testes. Biochemical alterations included elevated serum alkaline phosphatase, corticosterone, and interleukins-1, -2, and -3. Additional immune alterations included increased responsiveness of spleen cells to both thymus-dependent and thymus-independent mitogens and increases in the total number of B cells in the spleen. At doses not inducing weight loss, target organ effects included the appearance of plasma cells and infiltration of polymorphonuclear cells in lymph nodes; myeloid cell hypercellularity in bone marrow; hematopoiesis in spleen; subacute inflammation in forestomach; and periportal cytoplasmic vacuolization in liver. At the low doses, SMR-2 resulted in decreased responsiveness of spleen cells to mitogens and SMR-6 caused increased responsiveness. SMR-6 also increased interleukin-1 and-2 production at low doses. Biochemical effects included reduced activities of liver aryl hydrocarbon hydroxylase (AHH) and soluble brain protein kinase C. Overall, the results suggest that leukopoiesis and reduced liver AHH and reduced soluble protein kinase C activities are the primary and initial pharmacological and toxicological effects of retinoids.

The recent increase in the clinical use of synthetic vitamin A compounds has led to concer of possible side effects. Some of these effects are known to be influenced by dietary levels of vitamin K. We therefore compared the toxic effects of 13-cis-retinoic acid (13cisRA), retinyl acetate (ROAc), and N-(4-hydroxyphenyl)retinamide (4HPR) in male Sprague-Dawley rats maintained on diets containing different levels of vitamin K. Animals were fed either an NIH-07 diet supplemented with menadione (3.1 ppm vitamin K3), an NIH-07 diet not supplemented with menadione, or an AIN-076 purified diet devoid of vitamin K. The retinoids had no effect on prothrombin times of animals fed the supplemented diet. When menadione was omitted from the diet, however, 4HPR-dosed animals had elevated prothrombin times. This effect was observed as early as Day 7 and was accompanied by one confirmed hemorrhagic death. 13cisRA-dosed animals showed no change in prothrombin times. In the high-dose ROAc group, there was a twofold increase in prothrombin times but only after prolonged dosing. In animals fed the NIH-07 diets, 13cisRA and ROAc induced multiple bone fractures at all dose levels. In contrast, 4HPR administered at the highest dose induced only one fracture in one animal. Animals fed the purified diet lost weight faster and died sooner than those maintained on the other diets. Bone fractures were not observed in these animals because of early deaths resulting from hemorrhaging. For all retinoid-dosed groups maintained on the purified diet, changes in prothrombin times occurred as early as 1 week. The order of effect was 4HPR > ROAc > 13cisRA, with increases in prothrombin times correlating with increases in hemorrhagic deaths. Hence, the degree of retinoid-induced hemorrhage, but not the incidence of bone fractures, was inversely related to vitamin K levels in the diet. 13cisRA and ROAc, but not 4HPR, caused a dose-dependent reduction in plasma osteocalcin, an effect that correlated with retinoid-induced bone effects. In contrast, serum alkaline phosphatase was elevated in animals dosed with 13cisRA or 4HPR but not in those dosed with ROAc. For this enzyme, the electrophoretic pattern on agarose gel showed a decrease, compared to controls, in the major isozyme in serum of ROAc-dosed animals. Hence, plasma osteocalcin is a better predictor of retinoid-induced bone effects than serum alkaline phosphatase.

Menhaden oil, which has hypolipidemic and anticarcinogenic activity, reduces the hypertriglyceridemia caused by retinyl acetate. Male Sprague-Dawley rats were dosed daily for 30 days by gavage with either corn oil (CO); menhaden oil (MO); 20, 80, and 250 mg/kg retinyl acetate (ROAc) in CO; or 20, 80, or 250 mg/kg ROAc in MO. Hypertriglyceridemia by ROAc was reduced by coadministration of MO, and serum cholesterol values were reduced to levels similar to those for rats receiving MO alone. Coadministration of MO reduced the ROAc-induced fracture incidence at 80 mg/kg but not at 250 mg/kg. For groups dosed with ROAc and CO or MO, there were no differences in weightgain depression, elevation of serum alkaline phosphatase, or reduction of food consumption, suggesting that reduced absorption of ROAc was not the basis for the activity of MO. The reduction in retinoid toxicity by MO suggests a need for further study of the toxicity and anticarcinogenicity of retinoid/menhaden oil combinations.

In disposition studies, retinyl methyl ether (RME) was administered to rats in oral doses of 10 or 40 mg/kg. For the high dose, RME was eliminated from plasma with a terminal half-life of 19.5 hr but for the low dose the terminal phase could not be determined. For both doses, the concentrations of RME in the tissues examined (liver, spleen, adrenals, and mammary glands) were greater than those in plasma. In the adrenals of rats given the low dose, concentrations were as much as 10- to 100-fold higher. Concentrations of RME in the mammary gland, a site for chemopreventive activity, were also relatively high (about 1000 ng/g for the low dose and about 4000 ng/g for the high dose), and there was an elimination phase with a half-life of 63-81 hr. After administration of RME, the concentration of retinyl esters in the liver did not increase, and no retinyl esters were detected in the mammary gland. For toxicology studies, rats were administered 20, 40, and 80 mg/kg of RME or retinyl acetate (ROAc) daily for 28 days. The toxic effects of RME were similar to those of ROAc. At equivalent mg/kg doses, weight gain depressions, bone fractures, elevations in serum triglycerides, anemia, elevations in cholesterol in females, and reductions in serum albumin were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of these studies was to evaluate the short-term toxicity of theophylline, a compound present in tea and used in a variety of clinical applications. Fourteen-day repeated-dose toxicity studies were conducted in B6C3F1 mice and F344 rats of both sexes. Theophylline was administered in feed (0, 500, 1000, 2000, 4000, and 8000 ppm) or by gavage in corn oil (12.5-twice daily, 25, 50, 50-twice daily, 100, 200, 200-twice daily, and 400 mg/kg). Dosed-feed exposure to theophylline at concentrations up to 8000 ppm induced no significant toxicity except for dose-related uterine hypoplasia in rats. Palatability problems at that level precluded administration of higher concentrations. In the gavage study, 400 mg/kg was acutely toxic for both species, but mice and rats differed in that this same daily dose administered as two separate doses of 200 mg/kg was acutely toxic in rats but not in mice. No dose-related weight gain depression was evident in mice; weight gain was depressed in the majority of dose levels in rats and was pronounced at the higher levels. Clinical signs in mice were squinting and distended testes in males, and in rats, rapid respiration (all doses), squinting, and hunching. Gross necropsies, organ weights, clinical pathology, and pathology identified no target organs in mice, while histopathologic observations in rats suggested heart and stomach as possible target organs. Histopathologic effects in a number of other tissues, including lung, thymus, bone marrow, spleen, and uterus, were considered to reflect agonal changes in treated rats, possibly related to inanition. The results suggest that both species and sex differences exist with respect to sensitivity to theophylline toxicity, with F344 rats being more sensitive than B6C3F1 mice and male rats being more sensitive than female rats.

Sprague-Dawley rats were dosed by gavage daily for 28 days with 5, 15, or 50 mg/kg of N-(all-trans-retinoyl)-DL-leucine (RL), N-(all-trans-retinoyl)glycine (RG), or all-trans-retinoic acid (RA). On the basis of mortality incidence, fracture incidence, body weight, and histopathologic effects, RG was slightly to moderately less toxic than RA, and RL was significantly less toxic than RA or RG. Doses that had no effect on weight loss and produced no bone fractures were approximately 5 and 15 mg/kg/day for RA administered to males or females, respectively; greater than 15 mg/kg/day for RG administered to males or females; and greater than 50 mg/kg/day for RL administered to males or females. At these doses, RA and RG produced effects, detectable at the microscopic level, of lymphoid hyperplasia and hematopoietic cell proliferation in the spleen, lymphoid hyperplasia in lymph nodes, necrosis of the cortex of the thymus, hypertrophy of the zona fasciculata of the adrenal, a periportal pattern of cytoplasmic vacuolization in hepatocytes, hematopoietic cell proliferation in the liver, epithelial hyperplasia and subacute inflammation in the forestomach, and osteodystrophy. Serological alterations consisted of reduced serum albumin levels and elevated levels of triglycerides and alkaline phosphatase. For RL, similar microscopic effects, dependent on dose level and sex, were observed in spleen, thymus, adrenal, and liver. In vitro, RL was as active as RA in potentiating pokeweed mitogen-induced lymphocyte proliferation; RG was inactive. This study indicates that, relative to RA and RG, RL has less toxicity but similar immunological effects. Since RL and RG expressed little or no binding affinity for cellular RA-binding protein, the immunological effects of these retinoids may be expressed by mechanisms not linked to this protein.

Retinyl acetate, 13-cis-retinoic acid (13cisRA), and N-(4-hydroxyphenyl)-retinamide (4HPR) were assayed for their in vivo effects on hepatic levels of cytochrome P450, cytosolic glutathione-S-transferase, and quinone reductase. When given p.o. to Sprague-Dawley rats, all of the retinoids caused significant suppression in the levels of arylhydrocarbon hydroxylase, yet 13cisRA and 4HPR caused elevations in cytosolic levels of quinone reductase and glutathione-S-transferase, respectively. Scans of sodium dodecyl sulfate-polyacrylamide gels of microsomal proteins from the livers of retinoid-dosed animals showed changes in both the intensities and the number of stained bands. For microsomes from 13cisRA-dosed animals, there were additional changes in the absorption maximum of the carbon monoxide and octylamine difference spectra. There was, compared to controls, a 62% reduction in the NADPH-dependent binding of (+)-7S-trans-7,8-dihydro[7-14C]benzo(a)pyrene-7,8-diol to microsomal proteins from 13cisRA-dosed animals. Fluorography of the sodium dodecyl sulfate-polyacrylamide gels showed that the major reduction in metabolite binding occurred in the Mr 50,000 region of the gel. The reduction in the NADPH-dependent binding of (+)-7S-trans-7,8-dihydro[7-14C]benzo(a)pyrene-7,8-diol to microsomal proteins in vitro and the reduction in hepatic arylhydrocarbon hydroxylase levels correlated with a reduction in the in vivo binding of benzo(a)pyrene to rat liver DNA. Animals dosed for 7 days with 13cisRA, retinyl acetate, or 4HPR showed a 38, 27, and 40% reduction in binding of benzo(a)pyrene to liver DNA and a 29, 32, and 21% reduction in binding to stomach DNA, respectively, when the carcinogen was administered on the eighth day, and the tissues were harvested 24 h later. Binding to lung DNA was reduced by 23 and 11%, respectively, in the 13cisRA- and 4HPR-dosed rats. No differences were observed in binding to kidney. Thus, retinoids, by altering the metabolism of carcinogens, could influence the initiation stage of carcinogenesis.

Arotinoids, which are analogs of retinoic acid (RA) and retinol (RO) with the carbon skeleton in a rigid conformation, have more favorable therapeutic indices relative to all-trans-RA and all-trans-RO. The purpose of this investigation was to obtain preliminary in vivo toxicity data on SMR-2 (analog of RO) and SMR-6 (analog of RA), arotinoids with promising activity (ED50's of 20 × 10−11 and 5 × 10−11 m, respectively; ED50 of RA = 1 × 10−11 m) for reversal of keratinization in tracheal organ culture. A preliminary toxicity study was conducted in male B6D2F1 mice with gavage of retinoids in corn oil (0.01, 0.05, and 0.1 mg/kg/day of SMR-2 or SMR-6; 1, 5, and 10 mg/kg/day of RA as reference control). Due to lack of toxicity, each dose level for SMR-2 and SMR-6 was increased by 4-fold on Day 29 of dosing. The study was terminated on Day 57. Hypervitaminosis A (weight loss, alopecia, skin scaling, and bone thinning) was induced in the mid- and high-dose SMR groups; weight-gain depression was predominant in the high-dose RA group. The SMR compounds were approximately 100-fold more toxic, based on weight loss, than RA. In the SMR dose groups with hypervitaminosis A, white blood cell counts were elevated 2- to 4-fold; and there were microscopic lesions in skin, testes, epididymis, bone, thymus, bone marrow, peripheral lymph nodes, spleen, stomach, adrenal, and pituitary. The leukocytosis was attributed to leukopoiesis in spleen and bone marrow, which may be due to either a direct effect and/or a secondary response to a subacute inflammatory reaction in skin. Only peripheral lymph node hyperplasia was observed in SMR-2 and RA low-dose groups. Enlarged thymus, lymph node hyperplasia, leukopoiesis in spleen and bone marrow, elevated alkaline phosphatase with bone hypertrophy, and testicular degeneration were observed in the mid-dose RA group. The results indicate that immune stimulation may be a primary early response to retinoids and that skin, leukopoietic tissues, reproductive organs, stomach, and bone are primary targets for retinoid toxicity.