Antigen specific, suppressor cells induced by the immunomodulator FCE 20696 in aged NZB/WF1 mice

FCE 20696 is a new synthetic immunomodulating agent, active on some manifestations of the autoimmune disease of NZB/WF1 mice, in which a disorder of T suppressor cells (SC) has been described to be of pathogenetic relevance. The ability of FCE 20969 to induce SC in NZB/WF1 mice, which are then able to specifically inhibit the production of anti autologous red blood cells antibodies (aRBC Ab), was investigated. NZB/WF1 mice were chronically treated with the compound starting from the ninth week of age, sacrificed at different times and their spleen cells transferred to 9 weeks old, syngeneic mice. In donor animals SC were induced by rat RBC, whereas in the recipients the suppressive activity of transferred cells was evaluated from the appearance of aRBC Ab induced by the same antigen. The results show that antigen specific SC were present in both FCE 20696 treated and control young animals. In older controls, SC couldn't be induced, whereas in treated animals SC were present and able to transfer suppression into the recipients. FCE 20696 was active at 1.5 mg/Kg.     

Benzo(a)pyrene-induced early cytopathologic changes and peroxidase activity in the endometrium of ovariectomized rats

The present study was designed to investigate whether benzo(a)pyrene (BP) alone would have cytopathologic effects on the endometrium of ovariectomized rats, and whether DES can modulate or facilitate this cytopathology. The morphology and cytochemically detectable peroxidase activity in the endometrial epithelium of castrated rats were studied 24 hr after sc administration of three to six doses on consecutive days (one dose per day) either of corn oil, BP (50 mg/kg/day), DES (20 μg/kg/day), or a combination of the latter two agents. BP alone induced mitosis, peroxidase activity, and focal squamous cell metaplasia in the endometrial epithelium of castrated rats. Peroxidase activity was weak in the granular endoplasmic reticulum of the normal epithelial cells after three and six doses of BP. However, in the focal metaplasia, the superficial squamous cells contained strong peroxidase activity after six consecutive daily doses of BP. The focal metaplasia present only after six doses of BP consisted of three to four layers of squamous cells and one to two layers of basal cells. DES-treated animals showed no formation of metaplasia or structural alterations in the epithelium, but intense peroxidase activity was present in epithelial cells after three or six daily doses. The castrates treated with three or six combined doses of BP and DES showed significant ultrastructural changes in the epithelial cells. Vacuolization of the cisternae of the granular endoplasmic reticulum was observed in the epithelial cells after three doses of BP-DES. Distinct dilation and degranulation of the granular endoplasmic reticulum as well as enlargement of mitochondria were found in all the epithelial cells of the pseudostratified hyperplastic epithelium after six consecutive daily doses of the combination, BP-DES. These ultrastructural aberrations are clearly indicative of a cytotoxic effect of BP on the endometrial epithelial cells. Collectively these findings suggest that sc administration of BP induces peroxidase activity which may lead indirectly to lesions in the endometrium of ovariectomized rats, and DES may enhance the cytotoxicity of BP by a similar mechanism.     

Benzo[a]pyrene-induced necrosis in the HepG(2) cells via PARP-1 activation and NAD(+) depletion

Benzo[a]pyrene (BaP), a member of polycyclic aromatic hydrocarbons (PAH), has been reported to induce cell death in various cell types. However, the underlying mechanisms are controversial. In the present study, we report that BaP induces necrotic cell death in human hepatoma (HepG(2)) cells. The process is dependent on the activation of poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme responsible for repairing DNA damage. Once activated, PARP-1 catalyzes the formation of ADP-ribose polymers on acceptor proteins at the expense of NAD(+). Incubation of cells with high extracellular concentration of NAD(+) (5mM) after BaP treatment caused an elevation in intracellular NAD(+) level and blocked cell death. Inhibitor of PARP-1 suppressed both overactivation of PARP-1 activity and NAD(+) depletion. Moreover, addition of pyruvate (5mM), but not glutamate (5mM) or glutamine (5mM), could restore ATP production and prevent cell death. These results elucidated a sequence of events linking cellular metabolism to the progression of cell death induced by this organic toxicant.     

Anti-clastogenic effect of magnolol on benzo(a)pyrene-induced clastogenicity in mice.

It was previously reported that magnolol strongly inhibited the mutagenicity induced by the indirect mutagens [benzo(a)pyrene (B(a)P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-aminoanthracene (2AA), and 7,12-dimethylbenz[a]anthracene (DMBA)] in Salmonella typhimurium TA98 and TA100 in the Ames test, and that the mechanism of this anti-mutagenic effect may involve the inhibition of the metabolic activation of indirect mutagen enzymes. In this study, the in vivo anti-clastogenic effect of magnolol against clastogenicity induced by B(a)P was evaluated using the micronucleus test in mice. Animals were treated with an oral administration of magnolol (1, 10, and 100 mg/kg) at -24, 0, 24, 48, 72, and 96 h before a single intraperitoneal injection of B(a)P. Peripheral blood specimens were prepared 48 h after administration of B(a)P, and analyzed by the acridine orange (AO) technique. The results indicated that magnolol inhibited clastogenicity induced by B(a)P at various administration times. In order to elucidate the mechanism behind this effect, we measured the activity of the detoxifying enzymes [UDP-glucuronosyltransferase (UGT) and glutathione-S-transferase (GST)] and antioxidative enzymes [superoxide dismutase (SOD) and catalase] in the liver when treated with an oral administration of magnolol at various administration times. Its effect on clastogenicity created by exposure to oxidative DNA damage-inducing X-ray irradiation was also evaluated using the micronucleus test in mice. Results showed that magnolol increased the activity of both UGT and SOD enzymes, and also inhibited the clastogenicity induced by X-ray irradiation. Magnolol had an anti-clastogenic effect on B(a)P in the micronucleus test as well as an anti-mutagenic effect on indirect mutagens in the Ames test. The anti-clastogenic effect of magnolol was also suggested by the increases in UGT and SOD enzyme activity, and by the attenuation of oxidative damage induced by X-ray irradiation.     

Benzo(a)pyrene-induced acute neurotoxicity in the F-344 rat: role of oxidative stress

Given the link between neurotoxicity and exposure to pollutants, the potential behavioral neurotoxicity of benzo(a)pyrene [B(a)P] was investigated. Studies have established that B(a)P requires metabolic activation to highly reactive species to elicit many of its adverse effects. This study investigated the perturbation of nervous system function by correlating behavioral changes with the metabolism of B(a)P, antioxidant enzyme levels and lipid peroxidation in selected brain regions. The neurobehavioral effects of single oral doses of B(a)P (25-200 mg kg(-1) body weight) on motor activity were examined in male F-344 rats at 2, 4, 6, 12, 24, 48, 72 and 96 h post treatment. Parent B(a)P and metabolites were measured at the above mentioned time points by reverse phase HPLC. The activity of several antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and levels of malondialdehyde were determined at 6 and 96 h in both the striatum and hippocampus of B(a)P exposed rats. Suppression of motor activity (up to 70%) reached a maximum at 6 h, but was reversible at 96 h in all dose groups. The kinetics of disposition data show a strong link between B(a)P metabolism and the onset and duration of behavioral effects. Benzo(a)pyrene caused a 15-70% inhibition in the activity of superoxide dismutase and glutathione peroxidase and an enhancement in catalase and lipid peroxidation (up to 68%) in the striatum and hippocampus at 6 and 96 h post treatment, respectively. These findings suggest that B(a)P-induced acute neurobehavioral toxicity may occur through oxidative stress due to inhibition of the brain antioxidant scavenging system.     

Benzo(a)pyrene-induced metabolic responses in Manila clam Ruditapes philippinarum by proton nuclear magnetic resonance ((1)H NMR) based metabolomics

Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) which causes carcinogenic, teratogenic and mutagenic effects in various species and the level of contamination of this toxic agent in the marine environment is of great concern. In this study, metabolic responses induced by two doses (0.02 and 0.2 μM) of BaP were characterized in the gill tissues of Manila clam Ruditapes philippinarum after exposure for 24, 48 and 96 h. The high dose (0.2 μM) of BaP induced the disturbances in energy metabolism and osmotic regulation based on the metabolic biomarkers such as succinate, alanine, glucose, glycogen, branched chain amino acids, betaine, taurine, homarine, and dimethylamine in clam gills after 24 h of exposure. In addition, hormesis induced by BaP was found in clams exposed to both doses of BaP. Overall, our results demonstrated the applicability of metabolomics for the elucidation of toxicological effects of marine environmental contaminants in a selected bioindicator species such as the Manila clam.     

Benzo[a]pyrene-induced immunotoxicity in Japanese medaka (Oryzias latipes): relationship between lymphoid CYP1A activity and humoral immune suppression

Exposure to the environmental contaminant benzo[a]pyrene (BaP) results in suppression of immune function in both mammalian and fish species. This laboratory has previously demonstrated that a single intraperitoneal (IP) injection of BaP reduced lymphocyte proliferation, phagocyte-mediated superoxide generation, and antibody-forming cell (AFC) numbers in Japanese medaka (Oryzias latipes). The objective of the current study was to determine the role of BaP metabolism in the observed immunosuppression. Results from rodent studies have suggested that BaP elicits its immunotoxic effects via upregulation of cytochrome P4501A1 (CYP1A1) and the subsequent production of immunosuppressive BaP metabolites. In this study, exposure of medaka to 200 microg BaP/g BW significantly induced CYP1A expression or activity within lymphoid tissue 48 h post-IP injection; induction was observed specifically within distinct subpopulations of kidney mononuclear cells. Concurrent injection of fish with BaP and the CYP1A1 inhibitors alpha-naphthoflavone (ANF) or dehydroepiandrosterone (DHEA) resulted in inhibition of renal EROD activity and amelioration of BaP-induced suppression of medaka AFC numbers. Results of this study suggest that (1) BaP-induced suppression of medaka humoral immunity relies upon the CYP1A-catalyzed production of immunotoxic BaP metabolites and (2) BaP metabolites may be created in situ, directly by specific cells within kidney lymphoid tissue. Thus, apparently, mechanisms involved in BaP-induced immunosuppression have been phylogenetically conserved from fish to mammals.     

Cytoprotective effect of mangiferin on benzo(a)pyrene-induced lung carcinogenesis in swiss albino mice

Antioxidants are one of the key players in tumourigenesis, and several natural and synthetic antioxidants have been shown to have anticancer effects. In the present investigation, the efficacy of mangiferin on the antioxidant status of benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice was assessed. The animals were divided into five groups. The animals in groups I and V were normal control and mangiferin control, respectively. Groups II, III and IV were administered with benzo(a)pyrene (50 mg/kg body weight, orally) for 4 weeks (twice a week) to induced lung carcinogenesis. Starting 1 week prior to benzo(a)pyrene administration, group III animals were treated with mangiferin (100 mg/kg body weight) in the diet for 18 weeks; 12 weeks after benzo(a)pyrene administration, group III animals were treated with mangiferin that continued until the end of the experiment period (18 weeks). At the end of the experiment period, the reactive oxygen species, glutathione and the activities of antioxidant enzymes were assessed in both lung and liver tissues. The levels of glutathione, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, vitamin E and vitamin C were decreased in group II animals. However, in the mangiferin + benzo(a)pyrene-treated groups III and IV, the levels of GSH and the activities of antioxidant enzymes in both lung and liver were improved when compared with benzo(a)pyrene-induced group II animals. In addition, the finding that mangiferin decreased reactive oxygen species levels and enhanced antioxidant status suggests that this polyphenol might also be of value in the prevention of benzo(a)pyrene-induced lung carcinogenesis.     

Fisetin, a novel flavonol attenuates benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice

Lung cancer is the foremost cause of cancer mortality and is a growing economic burden worldwide. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a naturally occurring flavonoid is found in vegetables and fruits possesses anti-oxidative, anti-inflammatory and anti-proliferative effects in a wide variety of cancer. In the present study it is hypothesized that fisetin may provide chemopreventive as well as chemotherapeutic effects against experimental lung carcinogenesis. The present study was designed to investigate whether fisetin confers anti-cancer action against benzo(a)pyrene [B(a)P] induced lung carcinogenesis. Treatment with fisetin significantly reduced the degree of histological lesions, restored the levels of lipid peroxidation (LPO), enzymic and non-enzymic anti-oxidants in B(a)P-induced mice. Anti-proliferative efficacy of fisetin was assessed by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) in B(a)P induced mice showed increased PCNA expression which is restored upon fisetin administration. Together, our results depicts that fisetin can be used as chemopreventive agent against lung cancer.     

Immunopharmacological actions of lumin (II): Effect of lumin administration in NZB X NZW (B/W) F1 mice

Lumin was administered at doses of 0.1 to 100 micrograms/kg for 5 months to NZB/W F1 mice as the model animal for studying systemic lupus erythematosus (SLE), a human autoimmune disease. The increase in anti-thymic autoantibody level was significantly inhibited at doses of 1 to 100 micrograms/kg. Also, the induction of suppressor T cells by concanavalin A was significantly promoted. In addition, recovery activity was significantly observed, over-coming the reduction in plaque-forming cell (PFC) response of anti-sheep erythrocytes at a dose of 100 micrograms/kg, as well as the reduction in PFC response of anti-trinitrophenylated-lipopolysaccharide at doses of 0.1 to 100 micrograms/kg. The above results prove that lumin exhibits an immunomodulating effect against immune disease in mice.     

Effect of Sapthaparna (Alstonia scholaris Linn) in modulating the benzo(a)pyrene-induced forestomach carcinogenesis in mice

The chemopreventive effect of various doses of hydroalcoholic extract of Alstonia scholaris (ASE) was studied on the benzo(a)pyrene (BaP) induced forestomach carcinoma in female mice. The treatment of mice with different doses, i.e. 1, 2 and 4 mg/ml ASE in drinking water before, during and after the treatment with carcinogen, exhibited chemopreventive activity. The highest activity was observed for 4 mg/ml ASE, where the tumor incidence (93.33%) was reduced by 6.67%. Similarly, the tumor multiplicity reduced (61.29%) significantly (P<0.02) at 4 mg/ml in the pre-post-ASE treated group. However, the pre or post-treatment of mice with 4 mg/ml ASE did not show chemopreventive activity. These findings are corroborated by micronucleus assay, where treatment of mice with ASE before, during and after carcinogen treatment reduced the frequency of micronuclei (MN) in the splenocytes in a dose dependent manner. The MN frequency reached a nadir at 4 mg/ml ASE, the highest drug dose which showed maximum chemopreventive action. The ASE treatment not only reduced the frequency of splenocytes bearing one MN but also cells bearing multiple MN indicating the efficacy of ASE in inhibiting mutagenic changes induced by BaP. The pre or post-treatment of mice with 4 mg/ml ASE also significantly reduced the frequency of BaP-induced MN in the splenocytes of treated animals.     

Benzo[A]pyrene-induced oral carcinogenesis and chemoprevention: studies in bioengineered human tissue.

Oral cancer, originating from smoking-induced lesions of the basal cells in the complex stratified oral epithelium, is difficult to treat. Early detection of premalignant lesions, e.g., leukoplakia, has suggested the possibility of chemopreventive measures, such as topical application of antimutagenic/antiproliferative dietary or pharmaceutical agents. As an extension of a study in human oral epithelial cell monolayers, we determined the carcinogen, i.e., benzo[a]pyrene (BaP), transport, bioactivation, and DNA binding in a bioengineered human gingival epithelial tissue construct and the chemopreventive effects of dietary polyphenols. Short-term experiments showed that both types of compounds can traverse this tissue as well as be effectively taken up by the tissue. The model cigarette smoke carcinogen BaP very slowly, but to a great extent, accumulated in the tissue with maximal uptake at 24 h. Such exposure clearly resulted in DNA binding of BaP by the tissue. This DNA binding was associated with BaP-induced CYP1B1 as well as CYP1A1 expression, as evidenced by mRNA measurements. Cotreatment of the oral tissue with dietary polyphenols, including resveratrol and quercetin, and BaP, resulted in significant inhibition of the BaP-DNA binding. Using fluorescence microscopy as well as simultaneous autoradiography, we also demonstrated that quercetin indeed penetrates the entire stratified tissue layer, but that quercetin was also oxidized within the cells. Thus, this bioengineered oral tissue construct opens up improved ways of understanding and preventing/treating smoking-induced oral cancer.     

Differential protection by rat UDP-glucuronosyltransferase 1A7 against Benzo[a]pyrene-3,6-quinone- versus Benzo[a]pyrene-induced cytotoxic effects in human lymphoblastoid cells

UDP-glucuronosyltransferase 1A7 (UGT1A7) is a polyaromatic hydrocarbon (PAH)-inducible UGT with activity toward various benzo[a]pyrene (B[a]P) metabolites. To investigate the influence of rat UGT1A7 on B[a]P-induced cytotoxicity, human lymphoblastoid L3 cells were transfected with pMF6 (control expression vector), p167Dtk2 (microsomal epoxide hydrolase expression vector), or p167Dtk2-1A7 (epoxide hydrolase/UGT1A7 coexpression vector), and the cell populations were compared for sensitivity to B[a]P-induced effects. B[a]P inhibited cell proliferation and decreased relative cell survival of p167Dtk2 and p167Dtk2-1A7 cells to a similar extent. Metabolism studies using [(3)H]B[a]P revealed increased formation of glucuronide conjugates of B[a]P-4,5-diol, 3-OH-, or 9-OH-B[a]P and an unidentified metabolite by p167Dtk2-1A7 cells, but the presence of unconjugated metabolites suggested that glucuronidation capacity may be limited. No differences between p167Dtk2 and p167Dtk2-1A7 L3 cells were observed in the growth inhibitory effects of 3-OH-B[a]P or B[a]P-7,8-diol, but p167Dtk2-1A7-expressing cells were found to be less sensitive to B[a]P-3,6-quinone-induced effects on cell proliferation and relative cell survival. The effect was also observed in AHH-1 lymphoblastoid cells expressing UGT1A7 without epoxide hydrolase. The UGT1A7-expressing AHH-1 cells were also less sensitive to growth inhibition by B[a]P-1,6-quinone and B[a]P-6,12-quinone. Flow cytometric analysis of vehicle and B[a]P-3, 6-quinone-exposed cell populations showed an association between UGT1A7 expression and resistance to B[a]P-3,6-quinone-induced apoptosis and loss of cell viability. These data suggest that UGT1A7 may be preferentially active toward B[a]P-quinones and that UGT1A7 may represent the PAH-inducible UGT activity previously implicated in protection against toxic redox cycling by B[a]P-3,6-quinone.     

Cyclophosphamide and 15(S)-15 methyl PGE1 correct the T/B lymphocyte ratios of NZB/NZW mice

The lupus of NZB/NZW F1 female mice is associated with immune complex glomerulonephritis and premature death. Cyclophosphamide and 15(S)-15 methyl PGE1 therapy halt disease progression. Fluorescein conjugated antibodies were utilized to label specific leukocytes and the subsets were quantitated using a Fluorescence Activated Cell Sorter. Normal outbred CD-1 female mice showed a decrease in absolute T and B cell numbers with age, but the ratio of T and B cells remained essentially constant through 9 months of age. By contrast the NZB/W female mice showed decreased numbers of total lymphocytes relative to CD-1 controls at all ages. Moreover relative to CD-1s, there was a far greater decrease in T cell numbers (7 x for NZB/W versus 2 x for CD-1) and B cell numbers failed to decrease with age. The characteristic decline in T lymphocyte numbers and relative increase in B cell numbers in NZB/W mice were corrected with cyclophosphamide and PGE1 therapy. However, there was no selective modification of T cell subsets (L3T4+ or Ly2+) with therapy. Our investigation suggests correction of the abnormal T/B cell ratio may be a useful marker of therapeutic activity in NZB/W mice.     

Benzo(a)pyrene-induced cytochrome p4501A expression of four freshwater fishes (Oryzias latipes,Danio rerio,Cyprinus carpio,and Zacco platypus)

Oryzias latipes, Danio rerio, Cyprinus carpio, and Zacco platypus are useful indicator species for CYP1A biomarker studies; however, comparative studies have not been performed. To compare susceptibility, dose- and time-dependent CYP1A induction at the mRNA and protein levels in response to benzo(a)pyrene (BaP) exposure was analyzed. At the mRNA level, a statistically significant difference was found among the four species; however, such was not observed at the protein level. C. carpio showed the highest CYP1A induction level and the steepest slope in the dose–response curve. To assess susceptibility, the difference in CYP1A mRNA induction among species must be considered, and C. carpio was the most sensitive species of the four evaluated in terms of CYP1A expression.     

Effect of captopril treatment on proteinuria in NZB/NZW F1 hybrid mice

Oral treatment of female NZB/NZW F1 hybrid mice with captopril prevented the development of proteinuria and prolonged survival in mice demonstrating slight (trace to 1+) proteinuria. Captopril treatment also markedly reduced the incidence and magnitude of proteinuria and prevented death in mice showing significant (3+ or greater) proteinuria. Histopathological studies of the kidneys of treated mice further demonstrated improvements in the renal lesions of autoimmune mice. The mechanisms whereby captopril treatment influences the course of disease in NZB/NZW mice are not known.     

Treatment with cystamine reduces apoptosis in liver from NZB/W F1 mice

Increased population with hepatic diseases and apoptosis were found in patients with SLE and implicated in the pathogenesis of SLE. Since cystamine has been demonstrated to be beneficial to NZB/W F1 mice in our previous report, this study intends to investigate the effects of cystamine in liver from NZB/W F1 mice. Decreased apoptosis was detected in liver from NZB/W F1 mice given cystamine as compared to those given PBS by TUNEL and caspase-3 activity assay. Fas-dependent apoptotic proteins including Fas, cleaved caspase-8 and tBid were reduced in liver from NZB/W F1 mice given cystamine as compared to those given PBS. Additionally, the mitochondria-dependent apoptotic proteins including cytochrome c and Apaf-1 were reduced in liver from NZB/W F1 mice given cystamine as compared to those given PBS. Moreover, increased BCL-2 protein was observed in liver from both mice. Notably, increased NF-kappaB protein was detected in liver from NZB/W F1 mice given cystamine as compared to those given PBS. These experimental results suggest the effect of cystamine in reducing apoptosis in liver from NZB/W F1 mice through Fas-dependent and mitochondrial-dependent pathways. The phosphorylation of NF-kappaB (p65) could be a possible mechanism involving anti-apoptotic effects of cystamine in liver from NZB/W F1 mice.