Sulfasalazine exposure during pregnancy and lactation induces alterations in reproductive behavior in adult female rat offspring.

AIMS: Sulfasalazine (SAS) is the first line drug in the treatment of chronic inflammatory bowel diseases in pregnant women. SAS and its metabolites cross the placenta and can be transferred through the milk. However, the long-term consequences to the reproductive system of offspring from dams exposed to SAS have not yet been studied. Thus, our study investigated the effects of SAS treatment during gestational and lactational periods on maternal care in F0 and reproductive outcomes in F1 females. MAIN METHODS: Wistar female rats (n = 10/group) received 300 mg/kg/day of SAS dissolved in carboxymethyl cellulose (CMC), by gavage, from gestational day 0 to lactation day 21 and 3 mg/kg/day of folic acid during gestation. The control group received CMC only. On PND 21, the female pups were selected for reproductive evaluation at different time points: infancy and adulthood. The reproductive parameters evaluated were installation of puberty (vaginal opening and first estrus), estrous cyclicity, reproductive organs weight, histological analysis of the ovary follicles and uterus, analysis of oxidative stress in ovarian tissue, reproductive behavior (sexual and maternal), and fertility. KEY FINDINGS: SAS treatment decreased the retrieving behavior in F0 females. The F1 females presented an increase in the lordosis score, frequency of lordosis of magnitude 3, and lipid peroxidation of ovarian tissues in both infancy and adult life. SIGNIFICANCE: The SAS effects observed in the current study represent a relevant concern for public health, as they demonstrated that treatment with SAS compromised the maternal motivation of dams and induced reproductive alterations in F1 females.

Transcriptomic analysis reveals the mechanism of sulfasalazine-induced liver injury in mice.

Liver injury is one of the main toxic effect of sulfasalazine (SASP). However, the toxicological mechanism of SASP-induced liver injury remains unclear. In the present study, the liver injury was induced by orally treatment with SASP for 4 weeks in mice. The hepatic mRNA profiles were detected by RNA sequencing and the differentially expressed genes (DEGs) were analyzed by bioinformatics methods. The elevated serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin (TBIL), combined with the hepatic histopathological features verified that liver injury was successfully caused by SASP. Transcriptomic results showed that 187 genes (fold change > 1.5 and P < 0.05) were differentially expressed, of which 106 genes were up-regulated and 81 genes were down-regulated in SASP-treated group. Moreover, the further analysis showed that these 187 differentially expressed genes (DEGs) were enriched in 123 GO terms, which mainly including oxidation-reduction process, oxidoreductase activity and epoxygenase P450 pathway. KEGG pathway analysis showed 30 pathways including chemical carcinogenesis, retinol metabolism, arachidonic acid metabolism, linoleic acid metabolism and glutathione metabolism. Among these 187 DEGs, the top 22 hub genes were screened from network of protein-protein interaction (PPI) and verified by qRT-PCR. The results showed that the mRNA levels of hepatic drug-metabolizing enzymes, including cyp2b50, cyp2c50, cyp2c39, cyp2c38, cyp2c29, cyp2c54, cyp2c55, cyp2a5, gsta1, gsta2, gstt2, gstm2 and ephx1, were significantly up-regulated, while egfr and egr1 were down-regulated in SASP-treated group. Moreover, the mRNA levels of egfr and cyp2c55 exhibited a dose-dependent changes in SASP groups. Western blotting verified that the changes of protein levels of EGFR and CYP2C55 were consistent with mRNA levels. Considering that egfr has the highest score in PPI degree and cyp2c55 has the largest fold change in qPCR analysis, our present results suggested that the toxicological mechanisms of SASP-induced liver injury might be related to multi-biological processes and pathways, and egfr and cyp2c55 may play important roles in SASP-induced liver injury. The present study would be helpful for better understanding the hepatotoxic mechanism of SASP. However, the precise mechanism still needs further research.

Dual effects of sulfasalazine on rat sperm characteristics, spermatogenesis, and steroidogenesis in two experimental models.

There are reports of sulfasalazine (Salazosulfapyridine; SASP)-induced reproductive toxicity, but there it is not known whether the SASP molecule or its intestinal metabolites are responsible for this effect. Rats received SASP (150, 300, and 600mg/kg) for 60 consecutive days (in vivo). Additionally, epididymal sperm was isolated and incubated with SASP (10µM-1600µM) (in vitro). Markers of oxidative stress, mitochondrial function, and sperm functionality, along with testis histopathology as well as several steroidogenic genes and proteins, including steroidogenic acute regulatory (StAR) protein, cytochrome P450 side chain cleavage enzyme (P450scc; Cyp11a), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) were measured. SASP toxicity was evident as shown by severe testicular histopathological alterations, along with poor sperm parameters and increased markers of oxidative stress. Plasma testosterone level and steroidogenesis-related gene and protein (StAR, 3-beta-HSD, 17-beta-HSD) expressions, as well as mitochondrial membrane potential, were significantly decreased at high doses of SASP (in vivo). Interestingly, in vitro treatment of sperm with SASP not only caused no significant detrimental effect on rat sperm but also increased parameters of sperm functionality and decreased markers of oxidative stress. SASP had paradoxical actions on the rat sperm in these experimental models. The findings might be useful in understanding the mechanism(s) of SASP-induced reproductive toxicity. The present findings have opened a new molecular window into the relationship between disrupted steroidogenesis and mammalian reproduction indices and also are vital regarding clinical administration of SASP and human reproductive health.

Sulfasalazine induced oxidative stress: a possible mechanism of male infertility.

The mechanism of action of sulfasalazine (SASP) in male infertility is not well elucidated. For it, an oxidative stress-like mechanism inductor of infertility was hypothesized. Adult male Sprague-Dawley rats (20/group) were orally administered 0, 300, and 600mg SASP/kg body weight for 14 days. One-half of animals in each group remained an additional period of 14 days without treatment. SASP induced a significant decrease of superoxide dismutase (SOD) and glutathione reductase (GR) at the highest dose in both testis and epididymis. GR remained altered in these tissues within the recovery period. However, an increase in SOD was noted in epididymis. An increase in thiobarbituric acid-reactive substances (TBARS) was noted in all SASP-treated groups. In epididymis, catalase (CAT) significantly increased at 600mg/(kgday). These results suggest that SASP induces oxidative stress, which in turn might act as a possible mechanism of male-induced infertility.

Early changes in sperm motility, acrosome reaction, and gene expression of reproductive organs in rats treated with sulfasalazine.

Previously, we reported that decreased epididymal expression of CD59 and decay accelerating factor (DAF) genes may affect sperm motility and the acrosome reaction in rats treated long-term (28 days) with sulfasalazine. To investigate the early effects of sulfasalazine on the male reproductive tract, we presently examined sperm motility, the acrosome reaction, and gene expression in the testes and epididymides of rats treated with sulfasalazine for 1, 7 or 14 days. Reduced sperm motility and acrosome reactions were noted on day 7, however, there were no remarkable changes in testicular gene expression. On the other hand, attenuated epididymal gene expression of CD59 and DAF was observed as early as day 1. As CD59 and DAF are secreted from the epididymis and play a role in sperm maturation, we hypothesize that sulfasalazine affects sperm maturation as an early effect and that CD59 and DAF genes are related to the negative effect.

Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test.

A survey conducted as part of an International Workshop on Genotoxicity Testing (IWGT) has identified a number of compounds that appear to be more readily detected in vivo than in vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; effects on pharmacology, in particular folate depletion or receptor kinase inhibition. It is possible that at least some of these compounds are detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the 'correct' choice of test may not always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. A decision tree is outlined as a guide for the evaluation of compounds that appear to be genotoxic agents in vivo but not in vitro. The regulatory implications of these findings are discussed.

Effects of male reproductive toxicants on gene expression in rat testes.

Predictive biomarkers of testicular toxicity are needed for an efficient development of drugs. The purpose of the present study was to obtain further insight into the toxicity mechanisms of various male reproductive toxicants and to detect genomic biomarkers for rapid screening of testicular toxicity. Four reproductive toxicants, 2,5-hexanedione (Sertoli cells toxicant), ethylene glycol monomethyl ether (EGME; spermatocytes toxicant), cyclophosphamide (spermatogonia toxicant) and sulfasalazine, were orally administered to male rats once. Six hours after the single dosing, gene expression in the testes was monitored by cDNA microarray and real-time RT-PCR and the testes were histopathologically examined. No histopathological abnormality was detected except for slight degeneration of spermatocytes in the EGME-treated testes. cDNA microarray analysis revealed differential gene expression profiles, and it was possible based on the profiles to characterize the action of the compounds in the testes. Interestingly, 3 spermatogenesis-related genes -- heat shock protein 70-2, insulin growth factor binding protein 3 and glutathione S transferase pi -- were affected by all the compounds. The above changes of gene expression were detectable within a short period after the dosing prior to the appearance of obvious pathological changes. These data suggest that cDNA microarray is a useful technique for evaluation of primary testicular toxicity. Furthermore, we propose the above 3 spermatogenesis-related genes as potential biomarkers of testicular toxicity.

Protein expression analysis of rat testes induced testicular toxicity with several reproductive toxicants.

The utilization of safety biomarkers to predict the possibility of compound-related toxicity provides several advantages for drug discovery and development, especially at an early stage. The objectives of this study were to investigate the effects of male reproductive toxicants on protein expression profiles in the rat testes and to identify potential biomarker candidates. Four well-known reproductive toxicants, ethylene glycol monomethyl ether (EGME), cyclophosphamide (CP), sulfasalazine (SASP) and 2,5-hexanedione (2,5-HD), were administered to male rats in a single dose, and protein expression profiles were investigated after 24 hr by two-dimensional gel electrophoresis (2DE). Histopathological examination of the testes and serum concentration analysis were also performed. From the results of the comparison of 2D-gels among different doses of a compound and among compounds, 52, 20, 24 and 111 spots were nominated as differentially expressed spots with EGME, CP, SASP and 2,5-HD treatments, respectively. Several spermatogenesis-involved proteins were identified, including glutathione S-transferase (GST), testis-specific heat shock protein 70-2 (HSP70-2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphatidylethanolamine-binding protein (PEBP). Some of them were altered by more than one compound. In summary, remarkable histopathological findings were observed only in the EGME high-dose group, and most of the protein changes were detected before histopathological changes occurred. Therefore, the proteins identified in this study could potentially serve as biomarkers to evaluate male reproductive toxicity at an early stage of drug discovery and development.

Effects of sulfasalazine on sperm acrosome reaction and gene expression in the male reproductive organs of rats.

Sulfasalazine (SASP) has been reported to depress the fertility in men and experimental male animals, but the fundamental mechanisms of infertility caused by SASP are still unknown. This study was designed to investigate the mechanisms of infertility in rats treated with SASP at a dose of 600 mg/kg for 28 days, including monitoring of sperm motility using computer associated sperm analysis system and acrosome reaction by FITC-concanavalin A lectin staining. The sperm motility and acrosome reaction, which are important for fertilization, were significantly reduced by SASP. Furthermore, to investigate the molecular mechanisms of infertility induced by SASP, mRNA expression analysis in the testes was performed using cDNA microarray as a first screening. It was revealed that CD59, which is located on the acrosomal membrane and is known to be important for the reproductive function of sperm, was affected in the testes; this was also confirmed by real-time PCR analysis, but the spermatogenesis-related genes examined in this study were not affected. Therefore, we focused on CD59 and two other acrosome membrane related-genes: MCP and DAF. CD59, MCP, and DAF in the epididymides of SASP-treated rats were significantly decreased as assessed by real-time RT-PCR analysis and additionally, the expression of CD59 protein was found to be decreased by Western blotting. These results allowed us to hypothesize that the suppression of epididymal acrosomal membrane proteins synthesis with their consequent reduced incorporation to the sperm membrane leads to a depressed sperm motility and acrosome reaction, and thereby leads to infertility in SASP treated male rats.

In vitro and in vivo activity of the nuclear factor-kappaB inhibitor sulfasalazine in human glioblastomas.

Glioblastomas, the most common primary brain cancers, respond poorly to current treatment modalities and carry a dismal prognosis. In this study, we demonstrated that the transcription factor nuclear factor (NF)-kappaB is constitutively activated in glioblastoma surgical samples, primary cultures, and cell lines and promotes their growth and survival. Sulfasalazine, an anti-inflammatory drug that specifically inhibits the activation of NF-kappaB, blocked the cell cycle and induced apoptosis in several glioblastoma cell lines and primary cultures, as did gene therapy with a vector encoding a super-repressor of NF-kappaB. In vivo, sulfasalazine also significantly inhibited the growth of experimental human glioblastomas in nude mice brains. Given the documented safety of sulfasalazine in humans, these results may lead the way to a new class of glioma treatment.

Treatment of poor-prognosis early rheumatoid arthritis. A randomized study of treatment with methotrexate, cyclosporin A, and intraarticular corticosteroids compared with sulfasalazine alone.

OBJECTIVE: To determine whether a regimen of methotrexate, cyclosporin A, and corticosteroids introduced at onset in poor-prognosis rheumatoid arthritis (RA) can produce a significant improvement in outcome compared with standard monotherapy with sulfasalazine (SSZ). METHODS: Eighty-two consecutive patients presenting with new, untreated RA of less than 12 months' duration who fulfilled criteria for poor long-term outcome were randomized to receive either combination therapy (n = 40) or SSZ alone (n = 42). The primary outcome measures were remission and American College of Rheumatology (ACR) criteria for 20% improvement at 48 weeks. RESULTS: After 48 weeks, the numbers of patients who met the ACR criteria for 20% improvement were not significantly different between the two groups (combination 58% versus SSZ 45%), and similar numbers of patients had persisting clinical remission (approximately 10% both groups). During the first 3 months, there were significantly greater reductions in parameters of disease activity in the combination group. By 24 weeks, the swollen and tender joint counts, C-reactive protein levels, and erythrocyte sedimentation rates had fallen significantly in both groups, with a greater improvement in the swollen and tender joint count in the combination group. At 48 weeks, the radiographic damage score had increased by a median of 1 (range 0-42.5) in the combination group and 1.25 (range 0-72.5) in the SSZ group (P = 0.28; although there were significant differences in the scores for the right hand). There were significantly fewer withdrawals due to lack of efficacy in the combination group than in the SSZ group (1 of 40 versus 10 of 42; P = 0.007). In the combination group, dose reduction was needed in 22.5% because of hypertension and in 22.5% because of elevated creatinine levels. Over 48 weeks, serum creatinine increased in both groups, but particularly in the combination arm. CONCLUSION: In poor-prognosis RA patients, "aggressive" combination therapy led to more rapid disease suppression but did not result in significantly better ACR response or remission rates. This suggests that in poor-prognosis disease, an approach based on identifying patients with poor treatment responses before extra therapy is added ("step-up" approach) may be more appropriate than the use of combination therapy in all patients from the outset.

The in vitro immunomodulatory effects of sulfasalazine on human polymorphonuclear leukocytes, mononuclear cells, and cultured glomerular mesangial cells.

Sulfasalazine (SSA) was investigated for its effects on phagocytic activity of normal human polymorphonuclear neutrophils (PMN), proliferation of mononuclear cells (MNC) and cultured glomerular mesangial cells. At concentrations from 25 to 100 microM, it inhibited phagocytic activity of PMN and the 3H-thymidine incorporation of phytohemagglutinin (PHA)-stimulated human MNC in a dose-dependent manner. At comparable concentrations, sulfapyridine and 5-aminosalicylic acid, two of its major metabolites, did not show similar effects. SSA exhibited an inhibitory effect on both mouse and rat mesangial cells but at rather higher concentrations (0.5 mM). Excretion of interleukin (IL)-8 by lipopolysaccharide (LPS)-stimulated PMN was also markedly deterred in a dose-dependent manner but excretion of IL-8 by LPS-stimulated MNC was not interfered by SSA. Production of tumor necrosis factor (TNF)-alpha and IL-1beta by mouse mesangial cells was not blocked by SSA but production of IL-4 by these cells was inhibited by it (>0.1 mM). Inhibition of MNC was not due directly to cytotoxic effect of SSA on these cells as shown by fluorescein diacetate stain. Collectively, SSA inhibits phagocytosis and IL-8 excretion by PMN as well as mitogen-stimulated MNC reaction. On the other hand, at high concentrations, it inhibits glomerular mesangial cells and their IL-4 excretion but not TNF-alpha and IL-1beta excretion. These results can account for minimal nephrotoxic characteristic of SSA and suggest that it may be helpful in the treatment of immune-mediated glomerulonephritis.

Rat epididymal sperm motion changes induced by ethylene glycol monoethyl ether, sulfasalazine, and 2,5-hexandione.

Epididymal sperm was examined using the Hamilton-Thorne Sperm analyzer (HTM-IVOS, version 10.6) in male rats treated with known male reproductive toxicants that act by different mechanisms to detect effects on sperm motion. Three agents known to produce changes in sperm motion at high exposure levels were administered at lower levels. Ethylene glycol monoethyl ether (EGEE), sulfasalazine (SASP), and 2,5-hexandione (2,5-HD) were administered by oral gavage to adult male Sprague-Dawley rats at 250 or 500 mg/kg/day, at 300 or 600 mg/kg/day, or at 100 or 250 mg/kg/day, respectively. The males were treated with EGEE, SASP, and 2,5-HD for 35, 28, and 28 days, respectively. The males treated with EGEE and SASP were mated with untreated females to assess male fertility. All males were examined for body weight, testicular and epididymal weight, epididymal sperm count, and sperm motion. The sperm motion parameters included percentage of motile sperm, percentage of progressively motile sperm (progressive motility), curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), and straightness (STR). For the male rats treated with SASP, no treatment-related effects on percentages of motile sperm or sperm count were observed despite impaired male fertility. However, abnormal motion of epididymal sperm from the SASP treated males was detected by a significant reduction in mean progressive motility, VAP, and ALH, and an increase in BCF and STR. For the males treated with 2,5-HD for 4 weeks, most parameters generated by the HTM-IVOS indicated decreased sperm motion despite no remarkable changes in testicular weight, epididymal weight, or sperm count. In the EGEE-treated males at 250 mg/kg/day for 5 weeks, abnormal motion of epididymal sperm was detected by decreased progressive motility and increased BCF, although there were no treatment-related effects on testicular weight or male fertility. Progressive motility was decreased in all treated groups and the difference from the control value was of the greatest magnitude among the sperm motion parameters generated by the HTM-IVOS. Velocity parameters (VAP, VSL, VCL) responded sensitively to abnormal sperm motion in the SASP and 2,5-HD studies. In spite of decreased sperm motion, BCF values were significantly increased in all treated groups except the 7-week EGEE high-dose group, where there were no motile sperm to evaluate. ALH was significantly decreased in the treated groups in which remarkable effects on sperm motion were noted. There were no significant changes in ALH at the low-dose of EGEE at which only mild effects on sperm motion were observed. STR was increased for epididymal sperm from the males treated with SASP when compared with the controls. For the males treated with EGEE and 2,5-HD, however, STR was decreased when compared with the controls. There were no significant differences in LIN in any of the groups treated with SASP, in which remarkably reduced sperm motion was detected by the other parameters. In conclusion, among the parameters generated by the HTM-IVOS, progressive motility was significantly decreased in all treated groups and the most valuable for detecting slight changes in sperm motion induced by these three different target toxicants. Further investigation with a larger set of compounds is needed to evaluate which IVOS parameters are the most sensitive in detecting motion changes.

Extensive biliary excretion of the sulfasalazine analogue, susalimod, but different concentrations in the bile duct in various animal species correlating to species-specific hepatobiliary toxicity.

Studies on biliary concentrations of susalimod were conducted in rat, dog and monkey to clarify the interspecies differences observed in toxicology studies with respect to hepatobiliary toxicity after long-term administration of the compound. Dose-related bile duct hyperplasia appeared only in dogs at doses > or =75 mg/kg/day, while in rats and monkeys it did not appear at doses up to 1500 and 2000 mg/kg/day respectively. Biliary excretion was investigated after intraduodenal administration of susalimod in anaesthetised animals. In addition excretion routes were determined by collecting urine and faeces following a radiolabelled intravenous dose. Susalimod was extensively excreted via the bile in all animal species, > or =90%, mainly as non-conjugated parent compound. However, the local concentrations in bile varied between the species. Highest concentrations were obtained in the dog. The bile/plasma concentration ratio was 3400 in the dog, 300 in the monkey and 50 in the rat. In the dog, bile duct concentrations of susalimod about 30,000 micromol/l was obtained at plasma concentrations approximately similar to those at which hepatobiliary toxicity occurred, while in rat and monkey the levels were < or =7000 micromol/l at plasma concentrations similar to those obtained at the highest doses in the toxicology studies. From these results supported by a previous biliary excretion study in conscious dogs with chronic bile fistula receiving repeated administration of susalimod (Påhlman et al. 1999), it is likely that the hepatotoxic findings in dog are induced by the high concentrations of susalimod in the bile duct.

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.

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.

Short-term male reproductive toxicity study with sulfasalazine in the rat.

Sulfasalazine (2-hydroxy-5-[[4-[(2-pyridinylamino) sulfonyl] phenyl]azo]benzoic acid; SASP) was administered to rats in a short-term male reproductive toxicity study to further examine the utility of this grouping of techniques and to generate reference data with a substance that is known to cause reversible infertility in men. Adult male CD rats (10/group) were orally administered 0, 150, 300, or 600 mg SASP/kg body weight in divided doses for 14 d followed by a 2-week period without treatment. Males were killed on test day (TD) 15 or 29. At each time point, the reproductive system was evaluated by comparing testicular and epididymal weights, DNA ploidy distributions of testicular cell suspensions, testicular and epididymal histopathology, and epididymal sperm concentrations, motion, morphology, and breakage. Adding time as a factor in the protocol aids in distinguishing testicular from posttesticular effects. Changes in sperm quality after 2 weeks of test article administration (TD 15) predominantly reflect effects that occurred after the sperm entered the epididymis, while testicular effects predominated on TD 29. Beginning on TD 14, males to be killed on TD 29 were cohabited with untreated females (1:2). Females were killed at midgestation and examined for pregnancy status. Body weight gain was depressed in all SASP groups during the first 3 d of test article administration. Food consumption was depressed at the 300- and 600-mg/kg dose levels. No changes were seen in testicular weight, but epididymal weight was depressed at the 600-mg/kg dose level. DNA ploidy distributions determined by flow cytometry did not indicate that the kinetics of spermatogenesis were disturbed. However, alterations in sperm release, which have not previously been reported, were seen at all SASP dose levels. On TD 29, the percentage of progressively motile sperm was depressed and beat/cross frequency was increased at the 600-mg/kg dose level. No changes were observed in sperm morphology or breakage. Fertility was slightly depressed at the 600-mg/kg dose level. In this study, testicular histopathology provided the most sensitive endpoint for reproductive toxicity. The impairment of fertility immediately after treatment was stopped, when no changes were apparent in sperm release or sperm motion, suggested that decreased sperm concentrations and altered motility, while contributory, may not be the primary causes of SASP-mediated infertility.