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.

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.

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.

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.

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.

Direct and metabolism-dependent toxicity of sulphasalazine and its principal metabolites towards human erythrocytes and leucocytes.

1. The role of metabolites in sulphasalazine-mediated toxicity has been investigated in vitro by the use of human red blood cells and mononuclear leucocytes as target cells, with methaemoglobin formation and cytotoxicity respectively, being the defined toxic end-points. 2. Of the metabolites of sulphasalazine investigated, only sulphapyridine was bioactivated by human liver microsomes in the presence of NADPH to a metabolite which caused marked methaemoglobinaemia and a small, but statistically significant degree of mononuclear leucocyte cell death. 3. Methaemoglobinaemia was inhibited by ketoconazole but not by ascorbic acid (100 microM), glutathione (500 microM) and N-acetylcysteine (50 microM). In contrast, ascorbic acid and the thiols afforded complete protection for mononuclear leucocytes. 4. Sulphapyridine (100 microM) was converted in vitro to a metabolite (metabolite conversion 6.8 +/- 0.3%), the retention time of which on h.p.l.c. corresponded to synthetic sulphapyridine hydroxylamine. The half-life of sulphapyridine hydroxylamine in phosphate buffer (pH 7.4) was found to be 8.1 min. 5. In the absence of microsomes and NADPH, sulphapyridine hydroxylamine caused a concentration-dependent (10-500 microM) increase in methaemoglobinaemia (2.9%-24.4%) and cytotoxicity (5.4%-51.4%), whereas sulphasalazine, sulphapyridine, 5-hydroxy sulphapyridine and 5-aminosalicylic acid had no effect.

Evaluation of the mutagenicity of the anti-inflammatory drug salicylazosulfapyridine (SASP).

Salicylazosulfapyridine, commonly known as sulfasalazine or SASP, is an anti-inflammatory drug that is widely used in the treatment of diseases such as ulcerative colitis and Crohn's disease. Increases in sister chromatid exchanges (SCE) and micronuclei (MN) frequencies have been reported in lymphocytes of patients maintained on SASP therapy for up to 21 months. We have tested SASP for its ability to induce chromosome aberrations (ABS) and SCE in cultured Chinese hamster ovary (CHO) cells, ABS in mouse bone marrow cells, and MN in erythrocytes from both bone marrow and peripheral blood of mice. In vitro assays for ABS and SCE were negative. In vivo, SASP administered by single gavage at doses up to 1000 mg/kg did not increase ABS in bone marrow cells of male B6C3F1 mice; however, increases in MN were observed in the peripheral blood erythrocytes of male and female B6C3F1 mice administered 675, 1350 or 2700 mg/kg SASP by gavage for 90 days. Weak but significant dose-related increases in MN were also observed in the bone marrow cells of male B6C3F1 mice administered 500, 1000 and 2000 mg/kg SASP for 3 days. These positive findings in mice support the role of SASP in the induction of MN and SCE in humans, and suggest the need for further evaluation of possible adverse human health effects associated with SASP therapy.

Reversible male infertility due to sulphasalazine: studies in man and rat.

Sulphasalazine treatment for inflammatory bowel disease in man causes oligospermia, reduced sperm motility and an increased proportion of abnormal forms. On withdrawal of sulphasalazine these effects are found to be reversible and 15 pregnancies occurred at a median of 2.5 months after stopping sulphasalazine therapy. Seminal plasma concentrations of acid phosphatase fructose and PGE2 as well as the hormone profiles of patients on sulphasalazine for three months were found to be within normal limits. Sulphasalazine fed to male Sprague Dawley rats caused a dose dependent and reversible infertility with a significant reduction in litter size. Rats fed the metabolite sulphapyridine also had a reduced litter size when mated, while those fed the metabolite 5'aminosalicylic acid and a polymer of 5'aminosalicylic acid did not. It seems likely that the sulphapyridine moiety of sulphasalazine is responsible for the infertility seen, the effect being mediated at a late stage in sperm maturation.

Renal function was not impaired by treatment with 5-aminosalicylic acid in rats and man.

In rat experiments and a clinical trial we have examined the suspected nephrotoxic potential of 5-aminosalicylic acid (5-ASA), the biological active metabolite of sulfasalazine (SZ). Male Wistar rats were treated orally for 4 weeks daily with 30 and 200 mg 5-ASA/kg and 75 and 500 mg SZ/kg. The two renal marker enzymes N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30), alanineaminopeptidase (AAP; EC 3.4.11.2) and creatinine were monitored in urine. At the end of the experiment rats were sacrificed, the removed kidneys histologically examined and drugs, their metabolites and creatinine measured in plasma and urine. In 9 patients treated chronically for their Crohn's disease with 3 X 0.5 g 5-ASA daily in form of suppositories and an oral preparation urinary excretions of NAG, AAP and serum creatinine were also monitored before and during therapy. Neither the animal experiments nor the observations in patients gave any evidence of nephrotoxic lesions induced by 5-ASA. Thus, our data show that in the doses applied, 5-ASA was devoid of altering renal excretion in rats and man.

Increased sister-chromatid exchange frequency in patients receiving sulphasalazine therapy for ulcerative colitis.

Sister-chromatid exchange and micronucleus frequencies are reported for lymphocytes cultured in vitro from 15 patients receiving sulphasalazine therapy for ulcerative colitis and 15 controls matched for age and sex. While the patients did not differ from matched controls for micronucleus frequency there was a substantial rise in their sister-chromatid exchange frequency. This evidence of DNA damage is discussed in relation to the known cancer risk that ulcerative colitis carries and the possibility of an increased mutation rate in the germ cells of those receiving sulphasalazine therapy.