The role of toxicological interactions in chemical carcinogenesis

Toxicological interactions may occur not only when exposure to two or more chemicals is simultaneous, but also when exposures are separated in time. Often the sequence of exposure determines the nature of the toxicological response. This is illustrated with the two hindered phenolic antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). Evidence available suggests that in certain tissues exposure to BHT first and then to a carcinogen is without significant consequences, whereas exposure to BHT after a carcinogen may enhance tumor formation. On the other hand, exposure to BHA before carcinogen administration often has a protective effect, whereas exposure to BHA after a carcinogen sometimes protects and sometimes is without any influence. Mechanisms underlying these interactions may be induction of mixed function oxidases, production of cell hyperplasia or other, as yet ill-defined, events such as modification of biological reactivity.     

Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of moxifloxacin, a quinolone antibiotic

The chronic toxicity and carcinogenicity of Moxifloxacin (MOX), a bacterial gyrase-inhibiting fluoroquinolone antibiotic, were studied in male and female Wistar rats in an accelerated cancer bioassay (ACB). The ACB is a mechanistic initiation/promotion chronic toxicity and carcinogenicity study designed to assess potential carcinogenic activity of a test substance in critical organs in which human carcinogens are active. The organs studied were liver, lungs, urinary bladder, mammary gland, bone marrow, thymus, spleen and stomach. MOX was given daily by intragastric instillation at 500 mg/kg bw/day for the first 13 weeks to produce potential initiation, followed by promoters (PROs) for 24 weeks, or for the last 24 weeks after 13 weeks of exposure to initiators (INs). The INs, administered during the first 13 weeks, were diethylnitrosamine for the liver, N-n-butyl-N-(4-hydroxybutyl)nitrosamine for the urinary bladder, ethylnitrosourea for the hematolymphoreticular system, N-nitrosodimethylamine for lungs, methylnitrosourea for the stomach and 7,12-dimethylbenz(a)-anthracene for the mammary gland. The PROs, administered during the last 24 weeks after MOX, were phenobarbital for the liver, nitrilotriacetic acid for the urinary bladder, azathioprine for the bone marrow, butylated hydroxytoluene for the lung, butylated hydroxyanisole for the forestomach, and diethylstilbestrol for the mammary gland. The INs produced preneoplastic and neoplastic lesions which were not enhanced by MOX, and MOX plus PROs elicited no neoplastic effects, documenting that MOX did not produce either initiation or promotion of neoplasia in any of the target sites, or in any of the other twenty tissues examined.     

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.     

Forestomach lesions induced by butylated hydroxyanisole and ethylene dibromide: a scientific and regulatory perspective

Selected pathology lesions from 9 studies, 5 with butylated hydroxyanisole (BHA) and 4 with ethylene dibromide (EDB) are reviewed and their relative importance in regulatory evaluation is discussed. When Fischer 344 (F344) rats were fed BHA at 0.5% and 2.0% of the diet for 2 years, an increased number of rats of both sexes had epithelial hyperplasia of the forestomach at both treatment levels, compared to controls. At the 2.0% level, an increased number of rats had forestomach papilloma or forestomach squamous cell carcinoma. In a second study, in which F344 rats were fed BHA at 1.0% and 2.0% of the diet for 2 years, increased numbers of rats in both treatment groups were reported to have hyperplasia or papilloma of the forestomach. At the 2.0% level, increased numbers of rats developed squamous cell carcinoma of the forestomach. More Syrian golden hamsters fed BHA at 1.0% and 2.0% of the diet for 2 years reportedly had hyperplasia, papilloma or squamous cell carcinoma of the forestomach than did nontreated animals. Ingestion of BHA at 0.5% and 1.0% of the diet by B6C3F1 mice for 2 years was reported to produce an increase of animals with hyperplasia or papilloma of the forestomach at both dosage levels, compared to nontreated mice. When beagle dogs were fed BHA at 1.0% and 1.3% of the diet for 180 days, no lesions/tumors of the distal esophagus or stomach were identified at gross necropsy or by light or electron microscopy. When EDB was administered by gavage to Osborne-Mendel rats and B6C3F1 mice under conditions of the National Toxicology Bioassay Program, more rats and mice, both male and female, developed squamous cell carcinoma of the forestomach than did nontreated groups. EDB administered via inhalation to F344 rats and B6C3F1 mice did not cause squamous cell carcinoma of the forestomach; however, other neoplasms occurred which were considered to be treatment-related. Information gleaned from the BHA and EDB studies with multiple animal species facilitated regulatory decision-making regarding the potential toxicity/carcinogenicity of these compounds to man.     

Immunohistochemical cellular distribution of proteins related to M phase regulation in early proliferative lesions induced by tumor promotion in rat two-stage carcinogenesis models

We have previously reported that 28-day treatment with hepatocarcinogens increases liver cells expressing p21^Cip1, a G₁/S checkpoint protein, and M phase proteins, i.e., nuclear Cdc2, Aurora B, phosphorylated-Histone H3 (p-Histone H3) and heterochromatin protein 1α (HP1α), in rats. To examine the roles of these markers in the early stages of carcinogenesis, we investigated their cellular distribution in several carcinogenic target organs using rat two-stage carcinogenesis models. Promoting agents targeting the liver (piperonyl butoxide and methapyrilene hydrochloride), thyroid (sulfadimethoxine), urinary bladder (phenylethyl isothiocyanate), and forestomach and glandular stomach (catechol) were administered to rats after initiation treatment for the liver with N-diethylnitrosamine, thyroid with N-bis(2-hydroxypropyl)nitrosamine, urinary bladder with N-butyl-N-(4-hydroxybutyl)nitrosamine, and forestomach and glandular stomach with N-methyl-N′-nitro-N-nitrosoguanidine. Numbers of cells positive for nuclear Cdc2, Aurora B, p-Histone H3 and HP1α increased within preneoplastic lesions as determined by glutathione S-transferase placental form in the liver or phosphorylated p44/42 mitogen-activated protein kinase in the thyroid, and hyperplastic lesions having no known preneoplastic markers in the urinary bladder, forestomach and glandular stomach. Immunoreactive cells for p21^Cip1 were decreased within thyroid preneoplastic lesions; however, they were increased within liver preneoplastic lesions and hyperplastic lesions in other organs. These results suggest that M phase disruption commonly occur during the formation of preneoplastic lesions and hyperplastic lesions. Differences in the expression patterns of p21^Cip1 between thyroid preneoplastic and proliferative lesions in other organs may reflect differences in cell cycle regulation involving G₁/S checkpoint function between proliferative lesions in each organ.     

Antimutagenic/antioxidant activity of green tea components and related compounds.

The ability of green tea components and other antioxidant compounds to function as antimutagens/antioxidants has been well established, and their role in cancer prevention is supported by numerous epidemiological studies. We have utilized modified Ames tests, superoxide scavenging assays, and assays for protection against DNA scissions to compare and contrast the protective effects of various teas and commercial and laboratory-isolated tea components to those produced by compounds such as resveratrol, selenium, curcumin, vitamins C and E, quercetin dihydrate, sulforaphane, ellagic acid dihydrate, glutathione reduced, trolox, butylated hydroxanisole (BHA), butylated hydroxytoluene (BHT), and N-acetyl-L-cysteine (NAC). In Ames tests, employing hydrogen peroxide as a mutagen, epigallocatechin gallate (EGCG) produced the highest level of protection of all antioxidants tested. Measurement of protection against DNA scissions produced results that again showed that EGCG produced the strongest protective effects. In scavenging assays using a xanthine-xanthine oxidase (enzymatic system), epicatechin gallate (ECG) showed the highest scavenging potential. In a nonenzymatic (phenazine methosulfate-NADH) oxidizing system, EGCG once again showed the strongest effects. The implications of these and similar results are discussed in relation to cancer prevention and prevention of drug/antibiotic resistance.     

Rapid induction of forestomach tumors in partially hepatectomized Wistar rats given butylated hydroxyanisole

Rats subjected to two-thirds partial hepatectomy (PH) and given the antioxidant butylated hydroxyanisole (BHA) at a dietary concentration of 2% for 3 months developed forestomach lesions. Histologically, these lesions were classified as hyperplasia, dysplasia of the basal cell, papillomas, and carcinomas in situ. In intact rats forestomach carcinomas were seen by other investigators after feeding 2% BHA for 15-20 months. Histochemical studies of tumors revealed a marked increase in the phenotypic expression of the oncofetal enzyme, gamma-glutamyl transpeptidase (GGT) in the tumors of treated rats.     

The heterotopically transplanted rat urinary bladder as a model for detection of tumor-promoting urinary growth factor(s)

The heterotopically transplanted rat urinary bladder (HTB) was developed in our laboratory as a model to study the role of urine in urinary bladder carcinogenesis. With this model, normal urine was found to enhance urinary bladder carcinogenesis initiated by N-methyl-N-nitrosourea or N-butyl-N-(4-hydroxybutyl)nitrosamine. Two crude urinary components (Fractions I and II) were obtained by gel filtration chromatography; they stimulated ornithine decarboxylase (ODC) in a test bladder carcinoma cell line 804G, and promoted carcinogenesis in the HTB system. Fraction I was found to stimulate growth of 804G cells in vitro. Preliminary data indicate that Fraction I contains at least one, and possibly two heat-stable ODC-inducible and mitogenic components. Further characterization of these components is in progress. The HTB system has been demonstrated to be useful for other investigations; for example, alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC, when instilled repeatedly to the bladder lumen, inhibited tumorigenesis in HTBs.     

The effect of butylated hydroxyanisole and butylated hydroxytoluene on behavioral development of mice

The chronic ingestion of .5% butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) by pregnant mice and their offspring resulted in a variety of behavioral changes. Compared to controls, BHA-treated offspring showed increased exploration, decreased sleeping, decreased self-grooming, slower learning, and a decreased orientation reflex. BHT-treated offspring showed decreased sleeping, increased social and isolation-induced aggression, and a severe deficit in learning.     

Differential effects of nordihydroguaiaretic acid (NDGA) on B-cell subsets: reversal of NDGA-induced antibody suppression by cyclic GMP is subset specific

Nordihydroguaiaretic acid (4,4'-[2,3-dimethyl tetramethylene]-dipyrpcatechol) (NDGA), a reportedly specific lipoxygenase inhibitor, suppressed the in vitro murine plaque-forming cell (PFC) response to a thymus-dependent (TD) antigen, and the two subclasses of thymus-independent (TI) antigens, TI1 and TI2, at a final concentration of 33 microM. Suppression kinetics were inconsistent with those observed in previous experiments for other lipoxygenase inhibitors, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in that BHA and BHT exert suppression early in the 5-day PFC culture system, whereas NDGA suppressed through the 96th h of the 120 h culture period. The sulfhydryl-protective agent 2-mercaptoethanol (2ME) protected both the TD and TI responses. Dibutyryl cyclic GMP (dbcGMP) failed to restore NDGA-suppressed TD and TI1 PFC responses but restored the TI2 response when added at 0-, 24-, 48-, 72-, and 96-h at concentrations of 1-2 mM. NDGA inhibited lipopolysaccharide- (LPS-) induced increases in murine splenocyte cyclic GMP (cGMP) levels, and dbcGMP failed to accelerate the onset of the TI2 PFC response appreciably. The results of these and other laboratory studies indicated that NDGA may not be a specific inhibitor of lipoxygenase. Furthermore, the B-cell subset responding to TI2 antigens may be separated from the TD- and TI1-responding subsets because of the ability of dbcGMP to restore NDGA-suppressed TI2 responses but not the TD or TI1 response. The results suggest a fundamental difference in the biochemical pathways of B-cell subsets, and that cGMP metabolism in some cells may be linked to specific protein synthesis.     

Vascular damage and lipid peroxidation in choline-deficient rats

Dietary butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) protected rats against the vascular damage of acute choline deficiency. The protection was not due to decreased food intake or lower body weight in the animals fed the antioxidants. Dietary vitamin E supplementation did not protect against vascular damage. Free radical concentrations were higher in the liver, heart and kidneys of choline-deficient rats than in those of controls, but differences were small and variation was great. Thiobarbituric acid-reactive material (TBA) was consistently higher in livers of choline-deficient rats than in those of controls, but values were dependent on and inversely proportional to dietary levels of vitamin E. Conversely, TBA levels were high in livers of rats fed the BHA- and BHT-supplemented, choline-deficient diet. Therefore, hepatic TBA values did not always correlate with the extent of vascular damage. Diene conjugation was not detected in hepatic lipids of choline-deficient rats during the first 48 hr, but was found consistently after 10 days. The occurrence of diene conjugation in hepatic lipids was related directly to the incidence and severity of vascular damage.     

Butylated hydroxyanisole produces both mutagenic and desmutagenic derivatives under gastric conditions.

Dietary butylated hydroxyanisole (BHA) has been known to have inconsistent functions on carcinogenesis, both prevention and initiation. We assumed that both functions of BHA were introduced by the derivatives formed after the reaction with gastric components such as nitrite in the stomach. We then identified the derivatives produced by incubating BHA with sodium nitrite at pH 2.0 or pH 5.0. Eight derivatives were detected; 2-tert.-butyl-p-quinone (BQ), 3,3'-di-tert.-butyl-biphenyldiquinone-(2,5,2',5') (BBDQ), 2,6-di-tert.-butyl-8-hydroxy-dibenzofuran-1,4-quinone (BHDQ), 6-nitro-BHA, 2,2'-dihydroxy-3,3'-di-tert.-butyl-5,5'-dimethoxy-biphenyl (di-BHA), an oxidized product of di-BHA, and two unstable reaction intermediates. BQ was a major final product at pH 2.0, but not at pH 5.0. 6-Nitro-BHA and the oxidized products of di-BHA were also the final products. BBDQ was formed from di-BHA and easily converted to BHDQ. Their mutagenicity and desmutagenicity were assayed using Salmonella typhimurium strains. BQ and BBDQ were the mutagens of base-substitution type, BHDQ was the potent desmutagen against a mutagenicity of Trp-P-2, and the others had neither of the two activities. Thus, BHA was found to produce both the mutagen and desmutagen under the gastric conditions. BQ has been previously reported to be easily detoxified by glutathione, and BHA itself is well known to prevent carcinogenesis. In the assessment of dietary BHA on carcinogenesis, since one of the mutagens from BHA is easily detoxified in our bodies and another is converted to a desmutagen, BHA appears to be one of the favourable chemicals for us.     

BHA通过ERK信号转导途径促进小鼠胎肝细胞神经组织细胞特异基因表达的研究

目的：了解丁羟回香醚（BHA）对小鼠胎肝（FL）细胞神经组织特异基因表达的影响及其信号途径。 方法： 采用MACS试剂盒分离小鼠胚胎肝Sca-1+细胞，以DMEM+10%胎牛血清培养液培养；第4 d后，加入或者不加入细胞外信号调节蛋白激酶特异抑制剂PD98059 (25 μmol/L)处理24 h，再加入BHA处理24 h（0.2 mmol/L）。然后在无血清培养基中培养5 d。用Western blotting和半定量RT-PCR方法分析BHA处理前后基因表达。 结果： 经BHA诱导处理后，细胞表达神经组织细胞特异蛋白显著增加如神经丝轻链（NF-L）、神经丝重链（NF-H）、脑因子1（BF-1）和酪氨酸羟化酶（TH）。NF-L、NF-H、BF-1和TH分别增加6.32倍、2.73倍、3.37倍、2.68倍。而PD98059能明显抑制BHA诱导的神经组织细胞特异蛋白NF-L、NF-H、BF-1和TH的表达。 结论： BHA通过细胞外信号调节蛋白激酶途径促进小鼠FL Sca-1+细胞表达神经组织细胞特异抗原、结构和功能基因。     

Inhibitory effect of peptide vilon on the development of induced rat urinary bladder tumors in rats

The effect of peptide vilon (Lys-Glu) on urinary bladder carcinogenesis in rats was studied. Urinary bladder tumors were induced with a selective carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine. The tumors developed in 56% vilon-treated animals and in 75.5% controls. Vilon 2-fold decreased the incidence of preneoplastic and early neoplastic changes in urinary bladder mucosa and significantly inhibited carcinogenesis.     

Effects of oltipraz, BHA, ADT and cabbage on glutathione metabolism, DNA damage and lipid peroxidation in old mice

Eighteen-month-old female mice were fed defined diets for 2 weeks which contained 0.05% or 0.10% oltipraz, 0.10% anethole dithione (ADT), 0.10% butylated hydroxyanisole (BHA) or 20% lyophilized cabbage. All diets resulted in significant increases in hepatic reduced glutathione (GSH) content. Glutathione reductase and glutathione S-transferase activities were also significantly higher than the control values. All diets produced significant decreases in hepatic DNA damage (single strand breaks) and lipid peroxidation (malondialdehyde content). In general, similar effects were produced by the two dithiolthiones, oltipraz and ADT. More pronounced effects were produced by oltipraz and ADT than by BHA or cabbage in the diet. Diets high in antioxidants may be effective in retarding free radical reaction processes associated with aging and cancer.     

Influence of dosing vehicles on the preclinical pharmacokinetics of phenolic antioxidants

Phenolic antioxidants, such as butylated hydroxyanisole (BHA) and propyl gallate (PG), have demonstrated paradoxical cancer initiating and preventive actions in animals. Studies examining the disposition and biological effects of these agents have used solutions in ethanol-saline, PEG400-saline, corn oil, or DMSO. The aim of this study was to compare the pharmacokinetics of BHA and PG in mice following dosing in either a "control" dosing vehicle (ethanol-saline, 2:3) or a solution of an inclusion complex of each agent with hydroxypropyl-beta-cyclodextrin (HPB) in saline. Results demonstrate that BHA or PG are rapidly absorbed and eliminated in mice following i.p. or p.o. dosing in either dosing vehicle. Pharmacokinetic parameters of BHA estimated in mice correlated with those reported for other species, including humans ("Interspecies Scaling"), suggesting that exposures are proportional to body weight across species. Therefore, rodents are appropriate animal models to study these phenolic antioxidants. The oral absorption of PG was influenced by dosing vehicle in mice, suggesting the need for cautious selection of traditional nonaqueous vehicles (such as DMSO, ethanol, etc.) in the investigation of biological activities of these xenobiotics. Indeed, DMSO elevated plasma alanine aminotransferase (ALT) and alkaline phosphatase (ALP) concentrations following subchronic i.p. administration of various blank vehicles to mice. Such elevations in plasma concentrations of these enzymes are considered biomarkers of hepatotoxicity. The absolute oral bioavailability of PG (administered as an HPB complex) in rats was low (5%) suggesting extensive metabolism or incomplete absorption. The low oral bioavailability of these phenolic antioxidants in rodents suggests that the risk assessment of these antioxidants should include an evaluation of their metabolites as well.     

Changes in endogenous DNA damage in aging mice in response to butylated hydroxyanisole and oltipraz

The extent of DNA damage (single strand breaks) was measured in the livers of female Swiss-Webster mice up to 24 months of age. The effects of the antioxidants butylated hydroxyanisole (BHA) and oltipraz on this DNA damage were also measured. Oltipraz is an antioxidant which is structurally related to compounds found in cruciferous vegetables. From 6 months on the extent of DNA damage increased, reaching a maximum about 18 months old. Limited administration of both BHA and oltipraz to mice significantly reduced the levels of hepatic DNA damage.     

Effects of various bile acids and their sodium salts on development of pepsinogen-altered pyloric glands in rats.

Effects of dietary bile acids and their sodium salts on the development of pepsinogen-altered pyloric glands (PAPG) were examined in male WKY/N Crj rats initially given a single dose of 160 mg/kg body weight of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by gastric intubation. From week 3 the animals were administered basal diet containing 0.5% supplements of cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or their sodium salts (Na-C, Na-DC and Na-CDC), or 5% ascorbic acid (ASA) or its salt (Na-AS) for 18 weeks. The concentration of DCA and Na-DC was reduced to 0.3% from week 12. At week 20, animals were killed and the numbers of immunohistochemically-demonstrated PAPG were determined. Values were significantly higher with Na-C and Na-CDC than with the corresponding parent acids, and in the Na-C case PAPG development was greater than with MNNG alone. In addition, Na-CDC itself induced the numbers of PAPG significantly. These results suggest that bile salts are possible intrinsic promoters of gastric carcinogenesis. They were without effect, however, on forestomach lesions.     

Positive two-stage carcinogenesis in female sprague-dawley rats using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and 12-o-tetradecanoylphorbol-13-acetate (TPA) as promotor

Summary In contrast to a previous report by Shubik, the validity of the 2-stage skin carcinogenesis experiment was demonstrated in the rat. The modified experiment was carried out in female Sprague-Dawley rats using intragastrically administered DMBA as a carcinogen and the topically applied phorbol ester TPA as a promoter.Seven groups of animals were used. Two groups were treated with TPA only, two groups were initiated only with DMBA, two further groups were both initiated and promoted, and one group served as a control. Each of the initiated/promoted groups or only initiated or promoted groups contained one sub-group in which the animals had been bilaterally ovarectomized prior to the experiment.Hyperplasia of the dorsal epidermis occurred only in the promoted and in the initiated/promoted groups. Tumors of the back skin were observed exclusively after initiation/promotion. Ovarectomy — leading to a prolonged survival time of the animals — seems to be crucial for the manifestation of malignant skin tumors. Initiation/promotion also gives rise to tumors of the forestomach, the small intestine, the liver and the colon. Tumors in other organs (especially in the mammary gland and the Zymbal gland) were also be observed after initiation alone.Dedicated to Professor Wilhelm Doerr on the occasion of his 65th anniversary     

Immunosuppressive and anti-inflammatory action of antioxidants in rat autoimmune diabetes

Oxidative stress makes an important contribution to the development of autoimmune diabetes. We therefore tested the possible therapeutic value of two anti-oxidants, butylated hydroxyanisole (BHA) and pyrrolidine dithiocarbamate (PDTC), in the animal model of diabetes induced in susceptible DA rats by multiple low doses of streptozotocin (MLD-SZ, 20 mg/kg/day for 5 days). Administration of either BHA, or PDTC (50 mg/kg/day for 7 days), after finishing MLD-SZ injections, attenuated both the development of hyperglycemia and insulitis. Ex vivo analysis revealed that BHA treatment reduced the proliferation of autoreactive lymphocytes and down-regulated their adhesion to endothelium. In addition, BHA markedly attenuated the production of proinflammatory cytokines IL-1beta and TNF-alpha by both islets of pancreas and peritoneal macrophages. In parallel, macrophage release of cytotoxic oxygen and nitrogen intermediates superoxide anion (O(2)*(-)) and nitric oxide (NO*), respectively, was significantly inhibited. Finally, BHA treatment reduced intrapancreatic expression of inducible NO synthase (iNOS) and consequent production of NO* by pancreatic islets. Together, these data indicate that antioxidant agents might be a feasible therapeutic tools to interfere with development of autoimmune diabetes at multiple levels, including lymphocyte proliferation and adhesion, as well as the production of proinflammatory and cytotoxic mediators.