Characterization of mutagenic glucuronide formation from benzo(a)pyrene in the nonrecirculating perfused rat liver.

Excretion of mutagenic metabolites of benzo(a)pyrene into bile from livers of corn oil- or 3-methylcholanthrene-treated Sprague-Dawley rats perfused with a nonrecirculating perfusion system was quantitated. Mutagenic benzo(a)pyrene metabolites were detected using Salmonella typhimurium (strain TA 98) grown in the presence of limiting amounts of histidine. Microsomes were not included in the bacterial assay since metabolic activation was carried out by the perfused liver. Mutagenic activity was detected only if beta-glucuronidase was added to the assay mixture or if bile was treated with acid to hydrolyze glucuronides prior to assay. When livers were perfused with 20 microM benzo(a)pyrene, stable, mutagenic glucuronides were exported from corn oil-treated livers at maximal rates of 149 +/- 24 (S.E.) revertants/g/hr and at rates of 225 +/- 22 revertants/g/hr in livers from 3-methylcholanthrene-treated rats. Chromatography of bile by high-performance liquid chromatography demonstrated that two peak areas contained phenolic glucuronides which were hydrolyzed by beta-glucuronidase. These two peaks, one which cochromatographed with authentic 3-benzo(a)pyrenyl-beta-D-glucuronide, accounted for all of the mutagenic activity in bile from livers perfused with benzo(a)pyrene. A good correlation (r = 0.86) between rates of mutagen production and rates of formation of phenolic glucuronides was observed under a variety of experimental conditions. The mutagenic activity observed with pure 3-benzo(a)pyrenyl-beta-D-glucuronide exposed to beta-glucuronidase was 4 revertants/nmol. When the rate of mutagen production was divided by the rate of production of 3-benzo(a)pyrenyl-beta-D-glucuronide by the perfused liver, a value of 4 revertants/nmol was also obtained. Therefore, it is concluded that mutagens exported in bile from livers perfused with benzo(a)pyrene can be accounted for predominantly by hydrolysis products of phenolic glucuronides.

Effect of bile salts on rates of formation, accumulation, and export of mutagenic metabolites from benzo(a)pyrene produced by the perfused rat liver.

The effect of sodium taurocholate on the biliary export of stable mutagenic phenolic glucuronide metabolites of benzo(a)pyrene from livers of corn oil- or 3-methylcholanthrene-treated rats was studied using a nonrecirculating perfusion system. Sterile bile samples were collected every 4 min and assayed for mutagens using the Ames Salmonella (Ta 98) test without addition of microsomes but containing beta-glucuronidase. Rates of export of mutagens produced from benzo(a)pyrene (20 microM) into the bile were stimulated 5-fold by the bile salt sodium taurocholate, concomitant with a 2- to 3-fold increase in bile flow. Steady-state rates of 60 and 90 revertants/g/h were observed in bile when 20 microM benzo(a)pyrene was infused into livers from corn oil or 3-methylcholanthrene-treated rats, respectively. These rates of efflux were increased to 250 and 550 revertants/g/h by the addition of taurocholate. Rates of production of mutagenic phenolic metabolites which account for the mutagenic activity were determined by adding rates of efflux into bile and effluent perfusate with rates of accumulation of metabolites in the cell. In livers from 3-methylcholanthrene-treated rats, rates (8 min) of benzo(a)pyrene phenol formation averaged 300 nmol/g/h during the initial 20 min of perfusion but increased to 450 nmol/g/h after 1 h. The addition of taurocholate increased maximal rates of phenol efflux in the bile from 6 to 148 nmol/g/h and decreased rates of phenol accumulation in intracellular stores from 342 to 220. Rates of efflux into the vena cava effluent averaged 120 nmol/g/h and were not affected by taurocholate. Infusion of dehydrotaurocholate increased the appearance of metabolites of benzo(a)pyrene in the effluent perfusate but did not change rates of efflux into bile. Taurocholate doubled rates of output of phenolic metabolites into the effluent perfusate when bile flow was arrested by perfusion with calcium-free buffer. Thus, mutagenic glucuronides from benzo(a)pyrene phenols accumulated in hepatocytes much faster than rates at which they were exported. Total rates of production of phenolic glucuronides by the liver were not affected by bile salts; however, taurocholate stimulated their export into bile, while dehydrotaurocholate increased their concentration in the effluent perfusate. Both salts probably act by displacing metabolites from intracellular binding sites.

The growth hormone: insulin-like growth factor 1 axis is a mediator of diet restriction-induced inhibition of mononuclear cell leukemia in Fischer rats.

A leukemia cell transplant model and both in situ and in vitro bioassays were used to assess the roles of endogenous factors in mediating diet restriction (DR)-induced inhibition of mononuclear cell leukemia (MNCL) in Fischer 344 rats. DR-treated male rats (n = 35), which were fed 75% of ad libitum (AL) intake of NIH-07 open formula diet, had lower transplanted MNCL incidence (54 versus 77%; P = 0.039) with longer latency (P = 0.015) and decreased severity (P = 0.01) than AL-treated rats 12 weeks after inoculation with MNCL cells. Five-day proliferation rates of cultured MNCL (CRNK-16) cells in diffusion chambers implanted in DR-treated rats were 22% less than in AL-treated rats (P = 0.03), indicating that DR-dependent diffusible factor(s) modulate in situ MNCL cell growth. Serum from DR-treated rats supported lower in vitro CRNK-16 cell proliferation rates relative to serum from AL-treated rats. Serum levels of both growth hormone (GH) and insulin-like growth factor 1 (IGF-1) were over 50% lower in DR- versus AL-treated rats. An evaluation of the in vitro cell proliferative activity of a panel of purified factors showed that GH and IGF-1, but not 15 other growth factors, stimulated thymidine incorporation in CRNK-16 cells. Infusion of either GH or IGF-1 via osmotic minipumps restored in situ and in vitro CRNK-16 cell proliferation in DR-treated rats up to rates measured in AL-treated rats. Splenocytes from DR-treated rats, relative to AL-treated rats, were more sensitive to mitogen stimulation, displayed increased cell surface expression of receptors for class 1 and 2 major histocompatibility complex molecules, and were more cytotoxic to target tumor cells. Infusion of either GH or IGF-1 in DR-treated rats further enhanced mitogen responsiveness and natural cytotoxicity but reversed the DR-induced increase in major histocompatibility complex receptors. We conclude that DR modulates MNCL progression in Fischer 344 rats through both its influence on MNCL cell proliferation via suppression of the GH:IGF-1 axis and its enhancement of host defenses against tumor cells.

Insulin-like growth factor 1 (IGF-1) alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs.

In this study, we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates apoptosis in human breast cancer cells, HBL100, induced by diverse chemotherapeutic drugs. IGF-1 increased cell survival of HBL100 cells treated with 5-fluorouracil (antimetabolite), methotrexate (antimetabolite), tamoxifen (antiestrogen/antiproliferative), or camptothecin (topoisomerase 1 inhibitor) and after serum withdrawal. Elevated cell survival was not due to an increase in cell proliferation by IGF-1, but rather to an inhibition of apoptosis. Evidence for death by apoptosis was supported by cellular morphology and DNA fragmentation. There were no changes observed in Bcl-2 protein or bax mRNA levels. Extracellular matrix (ECM) is known to influence the apoptotic response of cells; therefore, the antiapoptotic effect of IGF-1 on breast cancer cells was examined using different ECMs: laminin, collagen IV, or Matrigel. IGF-1 protected cells from apoptosis induced by methotrexate on all ECMs tested, providing the first evidence that IGF-1 protects against apoptosis in three-dimensional culture systems. These data provide the rationale to search for drugs that lower serum IGF-1 in an effort to improve the efficacy of chemotherapeutic drugs for the treatment of breast cancer.

Effect of dietary protein concentration on yield of mutagenic metabolites from 1,2-dimethylhydrazine in mice.

The effects of varying dietary protein concentrations on the metabolism of 1,2-dimethylhydrazine (DMH) to mutagenic products by male C57BL/6 X C3H F mice were assayed by in vivo and in vitro methods. DMH and its metabolite, azoxymethane (AOM), did not increase the mutation frequency of Salmonella typhimurium (strain G-46) in vitro alone or in the presence of mouse liver homogenates capable of activating the promutagen dimethylnitrosamine. Methylazoxymethanol (MAM), another metabolite of DMH, was mutagenic in vitro without activation. S.c. administration of DMH, AOM, or MAM at dosages ranging from 0.2 to 0.8 mmol/kg of body weight caused dose-dependent increases in mutations of S. typhimurium in the host-mediated assay, and molar potencies increased progressively from DMH to AOM to MAM. S.c. or i.p. injections of AOM increased host-mediated mutagenesis within 20 min, while increases in mutagenesis by DMH required at least 1 hr. When [14C]DMH was administered, [14C]azomethane was expired immediately, while 14CO2 began to appear 1 hr after DMH administration. The percentage of administered [14C]DMH expired as azomethane varied inversely with dietary protein concentration, while AOM-induced host-mediated mutagenesis was directly proportional to dietary protein (p less than 0.01). The percentage of DMH converted to mutagenic end products was limited by losses of the volatile metabolite azomethane, especially in protein-deficient mice. Greater expiration of azomethane and decreased conversion of AOM to MAM, both seen with restriction of dietary protein, were associated with a smaller body burden of DMH metabolites.

Dietary restriction reduces insulin-like growth factor I levels, which modulates apoptosis, cell proliferation, and tumor progression in p53-deficient mice.

Diet contributes to over one-third of cancer deaths in the Western world, yet the factors in the diet that influence cancer are not elucidated. A reduction in caloric intake dramatically slows cancer progression in rodents, and this may be a major contribution to dietary effects on cancer. Insulin-like growth factor I (IGF-I) is lowered during dietary restriction (DR) in both humans and rats. Because IGF-I modulates cell proliferation, apoptosis, and tumorigenesis, the mechanisms behind the protective effects of DR may depend on the reduction of this multifaceted growth factor. To test this hypothesis, IGF-I was restored during DR to ascertain if lowering of IGF-I was central to slowing bladder cancer progression during DR. Heterozygous p53-deficient mice received a bladder carcinogen, p-cresidine, to induce preneoplasia. After confirmation of bladder urothelial preneoplasia, the mice were divided into three groups: (a) ad libitum; (b) 20% DR; and (c) 20% DR plus IGF-I (IGF-I/DR). Serum IGF-I was lowered 24% by DR but was completely restored in the IGF-I/DR-treated mice using recombinant IGF-I administered via osmotic minipumps. Although tumor progression was decreased by DR, restoration of IGF-I serum levels in DR-treated mice increased the stage of the cancers. Furthermore, IGF-I modulated tumor progression independent of changes in body weight. Rates of apoptosis in the preneoplastic lesions were 10 times higher in DR-treated mice compared to those in IGF/DR- and ad libitum-treated mice. Administration of IGF-I to DR-treated mice also stimulated cell proliferation 6-fold in hyperplastic foci. In conclusion, DR lowered IGF-I levels, thereby favoring apoptosis over cell proliferation and ultimately slowing tumor progression. This is the first mechanistic study demonstrating that IGF-I supplementation abrogates the protective effect of DR on neoplastic progression.

Expression of a prolactin-like factor in preneoplastic and neoplastic mouse mammary gland and cells.

Prolactin is a member of the growth hormone family and is required for the growth and terminal differentiation of the mammary gland. Ectopic production of this hormone has been reported in several species, including rat, sheep, goat and human mammary tissues. In this study, mouse mammary cell lines, xenographs in the mammary gland from these cell lines and from hyperplastic alveolar nodules, spontaneous tumours, and normal tissues were studied for de novo production of this growth factor. Prolactin transcripts were found by reverse transcriptase PCR in some neoplastic and preneoplastic tissues and in mouse mammary cell lines, NOG8 and CDNR4, but were not detected in the normal mouse mammary gland. Northern analysis revealed a 1 kb transcript for both cell lines that co-migrated with the prolactin pituitary transcript. Conditioned medium from NOG8 cells was positive for prolactin bioactivity by the Nb2 rat lymphoma cell proliferation assay, and Western analysis revealed the presence of immunoreactive proteins at M(r) 14,000 and 60,000. Prolactin-like bioactivity was not detected in conditioned medium from CDNR4 cells, but an immunoreactive protein of M(r) 60,000 was detected by Western analysis. The mouse mammary cell line, Comma D, was negative for prolactin transcripts; however, adenocarcinomas derived from inoculation of Comma D cells into the cleared mammary fat pad were positive by reverse transcriptase PCR in two of four cases. Hyperplastic outgrowths maintained in the cleared mammary fat pad as well as spontaneous tumors were positive for prolactin transcripts in one of four cases. These results suggest that prolactin can be produced ectopically by the neoplastic mouse mammary gland.

Attenuated allergic responses to house dust mite antigen in feed-restricted rats.

Caloric restriction has been shown to alter a broad range of immunological end points in both experimental animals and humans. The objective of this study was to investigate the effect of short-term moderate feed restriction (25% reduction) on allergic immune responses in Brown Norway rats. After 3 weeks of acclimation to their feed regimens, rats were sensitized and 2 weeks later challenged with house dust mite (HDM) antigen via intratracheal instillation. Feed restriction resulted in lower levels of antigen-specific IgE in serum and reduced antigen specific lymphoproliferative activity in pulmonary lymph nodes. Feed restriction also attenuated pulmonary inflammation, as evidenced by lower levels of lactate dehydrogenase and total protein, decreased infiltration of neutrophils and eosinophils, and reduced secretion of pro-inflammatory cytokine tumor necrosis factor (TNF)-[alpha] in bronchoalveolar lavage fluid. In addition, feed restriction decreased TNF-[alpha] secretion in serum and decreased mRNA expression of TNF-[alpha] and interleukin-6 in pulmonary lymph nodes. We conclude that feed restriction strongly dampened the allergic immune responses to HDM in rats and that this attenuation was associated with decreased expression and secretion of pro-inflammatory cytokines.

Comparative carcinogenicity of two structurally similar phenylenediamine dyes (HC blue no. 1 and HC blue no. 2) in F344/N rats and B6C3F1 mice.

Toxicology and carcinogenesis studies of 2 structurally-related p-phenylenediamines, HC Blue No. 1, and HC Blue No. 2 were conducted by administering each chemical in feed for 103 weeks to both sexes of Fischer 344/N rats and B6C3F1 (C57BL/6N x C3H/HEN) mice. Diets containing 0, 1500, or 3000 ppm HC Blue 1 were fed to male and female rats and male mice; female mice received diets with 0, 3000, or 6000 ppm. Diets containing 0, 5000, or 10,000 ppm HC Blue 2 were fed to male rats and mice and the females received diets containing 0, 10,000 or 20,000 ppm. These concentrations were compatible with long-term growth and survival. The results demonstrated substantial differences in the neoplastic and non-neoplastic lesions caused by these structural analogs. HC Blue 2 caused histocytosis in lungs and hyperostosis of the skull in rats, and splenic hematopoiesis, fibrous osteodystrophy, and hyperostosis of the skull in mice. These non-neoplastic lesions were not observed in rats or mice treated with HC Blue 1. Contrasting, in male and female mice, HC Blue 1 produced dose-related increases in the incidences of both adenomas and carcinomas of the liver. HC Blue 1 produced a marginally positive trend in hepatocellular nodules and carcinomas in male rats and dose-related increases in hyperplasias and neoplasms of the lungs in female rats. In contrast, there was no evidence of carcinogenicity for HC Blue 2 in either sex of rats or mice, despite the fact that it was administered 3-5 times the dose of the HC Blue 1. Since these 2 nitroaromatic compounds differ only in the methyl vs. 2-hydroxyethyl substituent on the secondary amine of ring carbon 4, the great discordance in their carcinogenicity is most probably due to side group-directed alteration in their metabolic profiles.

Isolation of the mutagenic and DNA adduct-inducing components from a commercial preparation of HC blue 1 using Salmonella (TA98) bioassay-directed HPLC fractionation.

In the present study we report the separation of the mutagenic impurities from the nitrophenylenediamine hair dye HC Blue 1. This was accomplished by bioassay-directed HPLC fractionation, using Salmonella strain TA98 and reverse phase HPLC analysis. The mutagenic fraction eluted between 80 and 90% methanol, whereas the HPLC fraction containing the parent compound HC Blue 1 eluted with 30% methanol and was non-mutagenic. 100% of the mutagenic activity applied to the column was recovered in fractions that did not possess the blue color of HC Blue 1. Also, HPLC-purified HC Blue 1 did not form DNA adducts (32P-postlabeling) in Salmonella strain TA98. On the other hand, commercial HC Blue 1 and the mutagenic fraction derived from commercial HC Blue 1 (HPLC-isolated) gave similar DNA-adduct profiles that consisted of 7 adducts. DNA adduction was examined concomitantly with mutagenicity and toxicity studies on the HC Blue 1 samples in TA98. The data indicated that, in Salmonella, both the mutagenicity and DNA adduction of commercial HC Blue 1 are due to impurities and not the parent compound.

Toxicity and carcinogenicity of hydroquinone in F344/N rats and B6C3F1 mice.

Toxicology and carcinogenesis studies were conducted by administering hydroquinone (more than 99% pure) by gavage to groups of F344/N rats and B6C3F1 mice of each sex for 14 days, 13 wk or 2 yr. 14-day studies were conducted by administering hydroquinone in corn oil to rats at doses ranging from 63 to 1000 mg/kg body weight and to mice at doses ranging from 31 to 500 mg/kg, 5 days/wk. In the 13-wk studies, doses for rats and mice ranged from 25 to 400 mg/kg. At those doses showing some indication of toxicity in the 14-day and 13-wk studies, the central nervous system, forestomach and liver were identified as target organs in both species and renal toxicity was observed in rats. Based on these results, 2-yr studies were conducted by administering 0, 25 or 50 mg hydroquinone/kg in deionized water by gavage to groups of 65 rats of each sex, 5 days/wk. Groups of 65 mice of each sex were given 0, 50 or 100 mg/kg on the same schedule. 10 rats and 10 mice from each group were killed and evaluated after 15 months. Mean body weights of high-dose male rats and high-dose mice were approx. 5-14% lower than those of controls during the second half of the study. No differences in survival were observed between dosed and control groups of rats or mice. Nearly all male rats and most female rats in all vehicle control and exposed groups had nephropathy, which was judged to be more severe in high-dose male rats. Hyperplasia of the renal pelvic transitional epithelium and renal cortical cysts were increased in male rats. Tubular cell hyperplasia of the kidney was seen in two high-dose male rats, and renal tubular adenomas were seen in 4/55 low-dose and 8/55 high-dose male rats; none was seen in vehicle controls or in female rats. Mononuclear cell leukaemia in female rats occurred with increased incidences in the dosed groups (vehicle control, 9/55; low dose, 15/55; high dose, 22/55). Compound-related lesions observed in the liver of high-dose male mice included anisokaryosis, syncytial alteration and basophilic foci. The incidences of hepatocellular neoplasms, primarily adenomas, were increased in dosed female mice (3/55; 16/55; 13/55). Follicular cell hyperplasia of the thyroid gland was increased in dosed mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicity and carcinogenicity of nitrofurazone in F344/N rats and B6C3F1 mice.

Toxicology and carcinogenesis studies were conducted by feeding diets containing nitrofurazone (99% pure) to groups of F344/N rats and B6C3F1 mice for 14 days, 13 wk or 2 yr. In the 14-day studies, in which doses ranged from 630 to 10,000 ppm, nitrofurazone was more toxic to mice than to rats. Accordingly, in the 13-wk studies, doses for rats ranged from 150 to 2500 ppm and for mice from 70 to 1250 ppm. At the higher doses, convulsive seizures and gonadal hypoplasia were observed in both species. Evidence of toxicity in rats also included degenerative arthropathy. For the 2-yr studies, rats were exposed to 0, 310 or 620 ppm nitrofurazone and the survival of male rats given 620 ppm was lower than that of controls (33/50, 30/50 and 20/50 in the control, 310- and 620-ppm groups, respectively). Nitrofurazone administration increased the incidences of mammary gland fibroadenomas in female rats (8/49, 36/50 and 36/50 in the control, 310- and 620-ppm groups, respectively). In male rats it was associated with a marginal increase in sebaceous gland adenomas and trichoepitheliomas of the skin, mesotheliomas of the tunica vaginalis, and tumours of the perputial gland. Nitrofurazone caused testicular degeneration (atrophy of germinal epithelium and aspermatogenesis) in rats, and degeneration of vertebral and knee articular cartilage in rats of both sexes. In mice, dietary concentrations of nitrofurazone for the 2-yr studies were 0, 150 or 310 ppm. In mice of each sex, nitrofurazone administration induced stimulus-sensitive convulsive seizures, primarily during the first year of study. In male mice, there was no evidence of any chemically-related carcinogenic effects, but there was a treatment-related decrease in survival (39/50, 31/50 and 27/50 in the control, 150- and 310-ppm groups, respectively). In female mice nitrofurazone induced ovarian lesions with increased incidences of benign mixed tumours (0/47, 17/50 and 20/50 in control, low- and high-dose groups, respectively) and granulosa cell tumours (1/47, 4/50 and 9/50 in control, low- and high-dose groups, respectively).

Fatty liver of growing rats fed excess lysine and its prevention by adenine or allopurinol.

Weanling male Sprague-Dawley rats were fed ad libitum 15% casein diets with and without 5.0% lysine-HCI, 0.25% adenine sulfate or 0.1% allopurinol for 2 weeks. Addition of lysine alone depressed 2-week growth from 94 to 65 g increased average daily urinary orotic acid excretion from 0.39 to 1.77 mg and increased the percentage of total liver lipids from 3.6 to 11.2. Adenine or allopurinol did not change growth but markedly enhanced lysine-induced orotic aciduria and completely prevented lysine-induced fatty livers. Reports by other show that adenine and allopurinol also prevent fatty livers or rats fed arginine-free diets or excess orotic acid. The authors conclude that lysine-induced orotic aciduria results from arginine deficiency caused by antagonism of arginine function by lysine, and that lysine-induced fatty liver probably results from a lesion identical to that produced by feeding excess orotic acid.

Roles for insulin-like growth factor-1 in mediating the anti-carcinogenic effects of caloric restriction.

This paper focuses on the role of insulin-like growth factor-1 (IGF-1) and its associated regulatory apparatus as a key endocrine, autocrine, and paracrine signalling system involved in mediating the anti-carcinogenic activity of dietary restriction. Literature is reviewed showing that the inhibitory action of dietary restriction on carcinogenesis is global and pervasive--it is effective in several laboratory species, for a variety of tumor types, and for both spontaneous tumors and tumors caused by different types of tumor-inducing agents. Evidence is presented showing the IGF-1 pathway responds appropriately to nutritional interventions including diet restriction. Recent evidence points to an obligatory role for the IGF-1 receptor in the establishment and maintenance of the transformed phenotype and reveals that IGF-1 in concert with insulin-like binding protein 3 and p53 is involved in autocrine/paracrine growth signaling pathways as adaptive responses to environmental stimuli. Considered together these works show that the IGF-1 pathway is uniquely poised to influence cellular transformation leading to the malignant phenotype by modulating the balance of cellular proliferation and cell death (apoptosis) in precancerous and cancerous cells and by influencing metastasis of nascent tumors. We evaluated these hypotheses directly using animal models of mononuclear cell leukemia, bladder transitional cell carcinogenesis, and breast cancer. Our studies demonstrate that manipulation of IGF-1 level through dietary intervention influences tumor growth and metastasis. Upregulation of this pathway demonstrated that increased IGF-1 stimulates tumor proliferation, progression and metastasis. Conversely, downregulation of this pathway in vivo as a consequence of dietary restriction results in antitumorigenic activity. We found that the functional disruption of IGF-1R markedly influences breast cancer metastasis in nude mice by suppressing cellular adhesion, invasion, and metastasis of breast cancer cells to the lung, lymph nodes, and lymph vessels. Epidemiological observations and clinical oncology results support the involvement of IGF-1 in carcinogenesis and anticarcinogenesis. This leads to the hypothesis that factors such as IGF-1 which regulate body size and composition may be related to human cancer incidence or prognosis. Additional understanding of this pathway and its interactions with other signaling pathways will advance our ability to develop new interventions towards decreased cancer risk in humans.

The sensitivity of the NTP bioassay for carcinogen hazard evaluation can be modulated by dietary restriction.

Studies were undertaken to compare outcomes when four chemicals were evaluated under typical NTP bioassay conditions as well as by protocols employing dietary restriction. Four chemicals, using three different routes of exposure (in utero [accomplished by feeding the dam dosed feed], dosed feed, and gavage) were used to 1) evaluate the effect of diet restriction on the sensitivity of the bioassay toward chemically-induced chronic toxicity and carcinogenicity; and 2) evaluate the effect of weight-matched control groups on the sensitivity of the bioassays. Control and chemical exposed F344 rats and B6C3F1 mice (50-60/group) were fed NIH-07 diet either ad libitum or at restricted levels such that body weights were approximately 80% of ad libitum control weights. The dietary restricted groups were either sacrificed at the end of two or 3-years. Results consistently show that feed restriction decreased the incidence of neoplastic and non-neoplastic lesions at a variety of anatomic sites in both control and chemical exposed animals. Furthermore, the sensitivity of the bioassay to detect chemical carcinogenic response were altered by dietary restriction: three of the four chemicals were found to increase the incidence of neoplastic lesions at four sites when evaluated under standard ad libitum conditions for 104 weeks. When unexposed and exposed groups were both subjected to dietary restriction, none of these 4 sites were detected as a target for carcinogenesis after two or three years. Rather, two different sites of carcinogenesis were detected. When the top dosed ad libitum fed animals were compared against their weight-matched control groups, a total of 10 sites were identified as targets for carcinogenesis. These included all four sites identified under the ad libitum protocol, both sites identified under the feed restricted protocol, and an additional four sites that were not identified under the other two protocols. These studies show that dietary restriction of all animals can be expected of decrease the sensitivity of carcinogenesis bioassays. However, restricting only unexposed groups (weight matching) of control for non-specific weight loss in chemical exposed groups yielded the most sensitivity among our comparisons.

Mechanistic studies on genotoxicity and carcinogenicity of salicylazosulfapyridine an anti-inflammatory medicine.

Salicylazosulfapyridine (SASP), which has been in clinical use for over 50 years, was reported by the National Toxicology Program to increase rat (F344 strain) urinary bladder and mouse (B6C3F1 hybrid) liver tumours under ad libitum (AL) feeding conditions, while under a feed restriction (FR) regimen, these tumours were not increased. The present investigations were undertaken to assess the implications of these results for the safety of SASP in humans. SASP and its 2 major metabolites, 5-aminosalicylic acid (ASA) and sulfapyridine (SP) were tested for in vivo induction of micronuclei in mouse bone marrow cells with or without prefolic treatment and for in vivo formation of DNA adducts in rat and mouse liver and urinary bladder. None exhibited mutagenicity or DNA reactivity. SASP and SP have induced sister chromatid exchanges and micronuclei (MN) in cultured human lymphocytes in the absence of liver activation enzymes and in B6C3F1 mice (but not in rats) MN in bone marrow and peripheral RBC. Treatment with folate reduces the frequency of MN. Perhaps the short (28 days) RBC lifespan in mouse underlies the sensitivity of this species. Thus, SASP without folate supplementation is an aneuploidogen. In a 2-year study in AL fed SASP-treated (high dose 337.5 mg/kg) rats, urinary pH was increased and urinary specific gravity was reduced at 60 weeks. At the end, this SASP group showed urothelial hyperplasia and papillomas in the urinary bladders of male rats primarily. In the FR high dose SASP group, the hyperplasia was reduced from 82% to 14%. At the end of 2 years, the incidence of multi-organ leukemia was reduced in both AL and FR high dose SASP groups. Thus, SASP caused intraluminal bladder changes in the rat (especially males) consisting of chronic urothelial stimulation, concretions, hyperplasia which resulted in neoplasia. In the mouse, because of species differences in liver ratios (mouse > rat) and, increasing (3 times higher) liver perfusion in the mouse, compared to the rat, there was hepatocellular toxicity and resulting preneoplasia and neoplasia within 2 years. These findings occurred in all AL SASP groups (flat curve without dose response); but were reduced under FR conditions. In this species, the multiorgan lymphoma incidence was reduced in both AL and FR high dose SASP groups. Thus, SASP and its major metabolites are not genotoxic. Folate deficiency associated with SASP administration is probably responsible for aneuploidy in lymphocytes and erythrocytes. SASP does not induce neoplasia directly in either livers, urinary bladders or other organs. Accordingly, SASP is judged to pose no carcinogenic risk to humans.

Urea synthesis from ammonia in periportal and pericentral regions of the liver lobule. Effect of oxygen.

Rates of urea synthesis were determined in periportal and pericentral regions of the liver lobule in perfused liver from fed, phenobarbital-treated rats by measuring the extra O2 consumed upon infusion of NH4Cl with miniature O2 electrodes and from decreases in NADPH fluorescence detected with micro-light-guides. Urea synthesis by the perfused rat liver supplemented with lactate (5 mM), ornithine (2 mM) and methionine sulfoximine (0.15 mM), an inhibitor of glutamine synthetase, was stimulated by stepwise infusion of NH4Cl at doses ranging from 0.24 mM to 3.0 mM. A good correlation (r = 0.92) between decreases in NADPH fluorescence and urea production was observed when the NH4Cl concentration was increased. Sublobular rates of O2 uptake were determined by placing miniature oxygen electrodes on periportal or pericentral regions of the lobule on the liver surface, stopping the flow and measuring decreases in oxygen tension. From such measurements local rates of O2 uptake were calculated in the presence and absence of NH4Cl and local rates of urea synthesis were calculated from the extra O2 consumed in the presence of NH4Cl and the stoichiometry between O2 uptake and urea formation. Rates of urea synthesis were also estimated from the fractional decrease in NADPH fluorescence, caused by NH4Cl infusion in each region, measured with micro-light-guides and the rate of urea synthesis by the whole organ. When perfusion was in the anterograde direction, maximal rates of urea synthesis, calculated from changes in fluorescence, were 177 +/- 31 mumol g-1 h-1 and 61 +/- 24 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, however, rates were 76 +/- 23 mumol g-1 h-1 in periportal areas and 152 +/- 19 mumol g-1 h-1 in pericentral regions. During perfusion in the anterograde direction, urea synthesis, calculated by changes in O2 uptake, was 307 +/- 76 mumol g-1 h-1 and 72 +/- 34 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, urea was synthesized at rates of 54 +/- 17 mumol g-1 h-1 and 387 +/- 99 mumol g-1 h-1 in periportal and pericentral regions, respectively. Thus, maximal rates of urea synthesis were dependent upon the direction of perfusion. In addition, rates of urea synthesis were elevated dramatically in periportal regions when the flow rate per gram liver was increased (e.g. 307 versus 177 mumol g-1 h-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Active transport of nitrofurantoin across the mammary epithelium in vivo.

Nitrofurantoin is a commonly used urinary tract antibiotic that has been found at high concentrations in human milk. In vivo studies in rats were carried out to determine the mechanism by which this drug crosses the mammary epithelium. Lactating rats were gavage-fed with nitrofurantoin, and their milk and plasma levels of the antibiotic were measured at intervals up to 8 hr. The average milk-to-plasma (M/P) ratio, calculated from the areas under the milk and plasma curves, respectively, was 23 compared with a ratio predicted to be about 0.3 on the basis of lipid partitioning and protein binding determinations. M/P ratios for two nitrofurantoin congeners were also calculated. The neutral compound furazolidone had a M/P ratio of about 1, as predicted, whereas the basic compound furaltadone had a M/P ratio of 3.49 compared with a predicted ratio of 1.4. These data suggest that nitrofurantoin and, to a lesser extent, furaltadone are actively transported across the mammary epithelium into milk.

Active transport of nitrofurantoin across a mouse mammary epithelial monolayer.

The antibiotic nitrofurantoin is transported against an electrochemical gradient into milk. A monolayer of CIT3 cells, a subline of the Comma 1D normal mouse mammary epithelial cell line, transports [14C]-nitrofurantoin against a concentration gradient from the basal to the apical solution when grown on membrane filters. In a side-by-side diffusion chamber with well-stirred solutions on both sides, the transfer rate is 50% higher in the basal-to-apical than in the apical-to-basal direction. Nonlabeled nitrofurantoin (500 microM) in the basal chamber equalized the transport in both directions, suggesting that a specific transporter is responsible for the basal-to-apical increment in flux. From inhibition studies, the apparent affinity of this transporter for nitrofurantoin is 50 microM. Changes in pH between 6.4 and 7.8 had no effect on the active transport component of the flux but did affect the passive flux component. Passive flux of the nonionized molecule was 2.6 times faster than that of the ionized molecule, but the ionized molecule did appear to cross the membrane passively. Our findings show that nitrofurantoin is actively transported across a mammary epithelial cell monolayer by a transporter whose affinity for nitrofurantoin does not depend on the anionic charge on nitrofurantoin. The pH dependence of a parallel passive pathway suggests that both nonionized and ionized forms of nitrofurantoin cross the membranes of the mammary epithelial cell by passive diffusion.