Exceptional activity of murine glutathione transferase A1-1 against (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-induced DNA damage in stably transfected cells

We have shown previously that the alpha class murine glutathione transferase (GST) isoenzyme mGSTA1-1, unlike other mammalian class alpha GSTs, is highly efficient in catalyzing the glutathione (GSH) conjugation of (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogenic metabolite of benzo[a]pyrene. The present studies were undertaken to determine the efficacy of mGSTA1-1 in cellular protection against (+)-anti-BPDE-induced DNA damage in HepG2 cells stably transfected with mGSTA1 cDNA. Untransfected HepG2 cells, vector-transfected HepG2 cells (HepG2-vector), and cells transfected with mGSTA4 cDNA (HepG2-mGSTA4), an alpha class murine GST isoenzyme with low (+)-anti-BPDE-GSH conjugating activity, were used as controls for comparison. Intracellular GSH conjugation of (+)-anti-BPDE was significantly higher in mGSTA1-1-overexpressing HepG2 cells (HepG2-mGSTA1) than in HepG2-vector or HepG2-mGSTA4 cells. The formation of DNA-adducts of (+)-anti-BPDE, following a 10-, 20-, or 30-min exposure to 0.1, 0.5, or 1.0 microM [3H](+)-anti-BPDE, was reduced significantly in cells transfected with mGSTA1-1 compared with HepG2-vector or untransfected HepG2 cells. Consistent with the results with purified protein, overexpression of mGSTA4-4 had no effect on (+)-anti-BPDE-induced DNA damage. The results of the present study indicated that mGSTA1-1 was exceptionally effective in affording protection against (+)-anti-BPDE-induced DNA damage in a cellular system.     

人细胞色素p450 IA2基因的构建及其功能检测──该基因在啮齿类细胞中代谢

本文首先构建了含人类细胞色素ｐ４５０ＩＡ２ｃＤＮＡ的ｐＺｉｐ－ｐ４５０ＩＡ２的真核表达载体，并分别将其转染到Ｖ７９细胞和原代大鼠肝上皮细胞中．Ｎｏｒｔｈｃｒｎ杂交和免疫组化实验显示，Ｖ７９转染细胞中有人ｐ４５０ＩＡ２的ＲＮＡ和蛋白水平的表达。细胞毒实验和ＨＧＰＲＴ位点突变率检测结果证明，外源性ｐ４５０ＩＡ２基因可在Ｖ７９细胞中代谢活化黄曲霉毒素Ｂ１．转染有ｐＺｉｐ－ｐ４５０ＩＡ２质粒的原代大鼠肝细胞可代谢活化５ｎｇ／ｍｌ的黄曲霉毒素Ｂ１．本研究说明了人ｐ４５０ＩＡ２ｃＤＮＡ在ＭＭＬＶ的５′ＬＴＲ系统驱动下，可在体外啮齿类细胞中代谢活化黄曲霉毒素Ｂ１．它为ＡＦＢ１致癌机制的研究提供了一个新的、更为有效的途径。     

Stable expression of rat sulfotransferase 1B1 in V79 cells: activation of benzylic alcohols to mutagens

We have constructed Chinese hamster V79-derived cell lines (V79-rSULT1B1-A and -B) that express rat sulfotransferase 1B1 (rSULT1B1). Sulfotransferase activity towards 1-naphthol was 1020 +/- 220 pmol/min/mg cytosolic protein in V79-rSULT1B1-A cells and 57 +/- 9 pmol/ min/mg in V79-rSULT1B1-B cells. These activities were similar over 100 population doublings and at varying cell densities. Immunostaining indicated a cytoplasmatic localization of rSULT1B1. Expression usually was homogeneous within colonies but showed some variation between colonies. The level of rSULT1B1 protein in V79-rSULT1B1-B cells was similar to that in rat liver but higher than in colon mucosa. The cytotoxicity of the benzylic alcohols 4H-cyclopenta[def]chrysen-4-ol and 6-hydroxymethylbenzo-[a]pyrene was enhanced >100-fold in V79-rSULT1B1-A cells compared with SULT-deficient cells (V79p). Likewise, these compounds showed mutagenic effects (at the hprt locus) in V79-rSULT1B1-A cells starting at a concentration of 0.02 and 0.01 micro M, respectively, but were inactive in V79p cells even at a concentration of 1 micro M. The cell line with the lower expression level, V79-rSULT1B1-B, showed only marginal toxification of the compounds investigated, indicating an important role of the expression level in the test system. A thoroughly characterized mammalian cell system, including positive controls, is now available for studying rSULT1B1-mediated bioactivation of promutagens and protoxicants.     

The development of a human cell line stably expressing human CYP3A4: role in the metabolic activation of aflatoxin B1 and comparison to CYP1A2 and CYP2A3

We have developed a human lymphoblastoid cell line, designated 3A4/Hol, which stably expresses human CYP3A4 cDNA. This cell line exhibited testosterone 6 beta-hydroxylase activity, produced immunologically detectable CYP3A4 protein and was more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 (AFB1) than was the parent cell line. The concentration-response for AFB1 cytotoxicity and mutagenicity in 3A4/Hol cells was compared to the responses of isogenic cell lines expressing comparable levels of human CYP1A2 (1A2/Hyg cells) and human CYP2A3 (2A3/Hyg cells). 1A2/Hyg cells were 3- to 6-fold more sensitive than 3A4/Hol cells to AFB1-induced mutation. 3A4/Hol cells were 10- to 15-fold more sensitive to AFB1-induced mutation than 2A3/Hyg cells. The differences in mutagenicity were supported by the relative binding of [3H]AFB1 to cellular DNA.     

Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450- expressing human liver cell lines

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.      

Modulatory effect of crocetin on aflatoxin B1 cytotoxicity and DNA adduct formation in C3H10T1/2 fibroblast cell

Crocetin is a carotenoid isolated from the seeds of Cape jasmine (Gardenia jasminoides). The cytotoxicity and DNA-adduct formation of rat microsome-activated aflatoxin B1 (AFB1) in the C3H10T1/2 cells were significantly inhibited by pretreatment of crocetin. Most significant inhibition was found at the time of 9 h after crocetin pretreatment. Under these experimental conditions, consistent elevation in the cytosolic glutathione (GSH) levels and the activities of GSH S-transferase (GST) and GSH-peroxidase (GSH-Px) were observed. Crocetin treatment also resulted in a decrease in AFB1-DNA adduct formation in vitro, while no effect of crocetin on the formation of AFB1-8,9-oxide in vitro system was detected as measured by the Trisdiol method. From these results, we suggested that the protective effect of crocetin on the AFB1-cytotoxicity in C3H10T1/2 cells might be due to the cellular defense mechanisms that elevated the cytosol GSH and the activities of GST and GSH-Px.     

Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.     

Glutathione conjugation of microsome-mediated and synthetic aflatoxin B1-8,9-oxide by purified glutathione S-transferases from rats.

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B1 (AFB1)-epoxide and styrene oxide has been investigated with purified GSH S-transferases (GSTs) from rats. Both styrene oxide and AFB1-epoxide were conjugated preferentially by millimicrons GSTs 3-3, 3-4 and 4-4 as compared to alpha GSTs 1-1, 1-2 and 2-2. The highest catalytic activity with styrene oxide conjugation was associated with GST 4-4. The highest catalytic activity with microsome-mediated AFB1-epoxide conjugation was observed with GST 3-3 whereas with the synthetic AFB1-epoxide conjugation was seen with GST 4-4. The catalytic activity of pi GST 7-7 was intermediate to millimicrons and alpha GSTs. It is suggested that GST 3-3 may play an important role in inactivation of AFB1-epoxide generated in vivo in the rat.     

Sister chromatid exchanges and chromosome aberrations in V79 cells induced by aflatoxin B1, B2, G1 and G2 with or without metabolic activation

Cells from Chinese hamster cell line V79 were pulse treated for one hour with aflatoxin (AF) B1, B2, G1 and G2 at various doses with or without the metabolic activation system-S9 mix. With metabolic activation, all these toxins caused significant increases of frequencies of sister chromatid exchanges (SCE) in a dose-dependent manner. Based on the rate of SCE induction, the relative order of potency was established as AFB1 > G1 > G2 > B2. Without metabolic activation, all four toxins had no or very little effect over the same dose ranges used for SCE induction with activation. Induction of chromosome aberrations in V79 cells after a one hour pulse treatment with AFB1 and G1 plus S9 mix was also studied. A dose and time dependent increase of aberrations was observed for both compounds. AFB1 again is a more potent aberration inducer than AFG1.     

Suppression of peritoneal metastasis in human gastric carcinoma by enhanced immunogenicity of B7-1 transfection

B7-1, a co-stimulatory factor, has been reported to induce cytotoxic T lymphocytes (CTL). In the present study, we transfected B7-1 genes into a gastric cancer cell line (2MD3) and analyzed the effects of B7-1 transduction on peritoneal metastasis in vitro and in vivo. We revealed that mononuclear lymphocytes show significantly stronger adherence and cytotoxicity to B7-1 transfected cells (2MD3/B7) than to their parent 2MD3 cells. We also demonstrated that mice inoculated with 2MD3/B7 cells in the peritoneal cavity have a significantly better survival rate than those inoculated with 2MD3 cells (log-rank test, p<0.01). Histologic findings showed that leukocytes intensively infiltrate the 2MD3/B7 metastatic nodules, but can scarcely be observed in the nodules associated with 2MD3 cells. These findings indicate that the B7-1 may play an important role in suppressing peritoneal metastasis by the mechanism of enhanced immunogenicity, and that B7-1 gene transduction might be effective against peritoneal metastases of gastric cancer.     

Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B(1) in human lung: differential cellular distribution and lack of significance of the GSTM1 genetic polymorphism.

Epidemiological studies suggest that aflatoxin B(1) (AFB(1)), a mycotoxin produced by certain Aspergillus species, may play a role in human respiratory cancers in occupationally-exposed individuals. AFB(1) requires bioactivation to the corresponding exo-8,9-epoxide for carcinogenicity, and glutathione S-transferase (GST)-catalyzed conjugation of the epoxide with glutathione (GSH) is a critical determinant of susceptibility to AFB(1). Of the purified human GST enzymes studied, the polymorphic hGSTM1-1 has the highest activity towards AFB(1) exo-epoxide. The influence of the GSTM1 polymorphism on AFB(1)-GSH formation, as well as the abilities of cytosols from preparations enriched in different isolated lung cell types to conjugate AFB(1)-epoxides, were examined. In whole-lung cytosols from patients undergoing clinically indicated lobectomy, GSTM1 genotype correlated with GSTM1 phenotype as determined by [(3)H]trans-stilbene oxide conjugation: GSTM1-positive = 295 +/- 31 pmol/mg/h (n = 6); GSTM1-negative = 92.8 +/- 23.3 pmol/mg/h (n = 4) (P < 0.05). In contrast, conjugation of microsome-generated [(3)H]AFB(1)-epoxides with GSH was low and variable between patients, and did not correlate with GSTM1 genotype: GSTM1-positive = 11.9 +/- 8.1, 111 +/- 66 and 510 +/- 248 fmol/mg/h (n = 6); GSTM1-negative = 15.3 +/- 16.7, 167 +/- 225 and 540 +/- 618 fmol/mg/h (n = 4) (for 1, 10 and 100 microM [(3)H]AFB(1), respectively). GSH conjugates of AFB(1) exo-epoxide and the much less mutagenic stereoisomer AFB(1) endo-epoxide were produced in a ratio of approximately 1:1 in cytosols from both whole lung and isolated cells. Total cytosolic AFB(1)-epoxide conjugation was significantly higher in fractions enriched in alveolar type II cells (3.07 +/- 1.61 pmol/mg/h) than in unseparated lung cells (0.143 +/- 0.055 pmol/mg/h) or fractions enriched in alveolar macrophages (0. 904 +/- 0.319 pmol/mg/h; n = 4) (P < 0.05). Furthermore, AFB(1)-GSH formation and percentage of alveolar type II cells in different cell fractions were correlated (r = 0.78, P < 0.05). These results demonstrate that human lung GSTs exhibit very low conjugation activity for both AFB(1)-8,9-epoxide stereoisomers, and that this activity is heterogeneously distributed among cell types, with alveolar type II cells exhibiting relatively high activity. Of the GSTs present in human peripheral lung which contribute to AFB(1) exo- and endo-epoxide detoxification, hGSTM1-1 appears to play at most only a minor role.     

Effects of BTG2 on proliferation inhibition and anti-invasion in human lung cancer cells

The objective of the study was to investigate the impact of the B cell translocation gene 2 (BTG2) on lung cancer cell growth, proliferation, metastasis, and other biological characteristics and to provide experimental evidence for the biological treatment of human lung cancer. A pcDNA3.1-BTG2 eukaryotic expression vector was constructed and transfected into the human lung cancer cell line A549. The biological changes in the BTG2-expressing cells were analyzed using growth curves, the MTT (tetrazolium) assay, propidium iodide (PI) staining, and the Transwell invasion chamber. Additionally, Western blotting was used to determine the impact of BTG2 on the protein expression of cyclin D1, MMP-1, and MMP-2. Compared to the empty vector-transfected A549 cells or the mock-transfected A549 cells, the pcDNA3.1-BTG2-transfected A549 cells grew significantly slower. No significant differences were detected between the empty vector-transfected group and the mock-transfected A549 cells. The growth curve analysis and the PI staining showed that the pcDNA3.1-BTG2-transfected cells grew significantly slower than the empty vector-transfected A549 cells (P < 0.05). The cell invasion assay results suggested that the invasion rate of the pcDNA3.1-BTG2-transfected A549 cells was significantly slower than the invasion rate of the empty vector-transfected group and the mock-transfected group (P < 0.05). The overexpression of BTG2 may inhibit the protein expression of cyclin D1, MMP-1, and MMP-2 in A549 cells. The overexpression of BTG2 may inhibit the growth, proliferation, and invasiveness of the A549 human lung cancer cell line.     

Mutation of Chinese Hamster V79 cells and transformation and mutation of mouse fibroblast C3H/10T1/2 clone 8 cells by aflatoxin B1 and four other furocoumarins isolated from two Nigerian medicinal plants

Mutation by aflatoxin B1 (AFB1), imperatorin, marmesin, chalepin, and 8-methoxypsoralen (MOP), with and without black light (BL; long-wavelength ultraviolet light) activation, was determined at the hypoxanthine-guanine phosphoribosyltransferase locus (8-azaguanine resistance) in Chinese hamster V79 cells and at the ouabain locus in mouse C3H/1OT1/2 cells. Transformation by these furocoumarins under the same activation conditions was also investigated in C3H/1OT1/2 cells. In V79 cells, AFB1 induced a 4-fold maximum mutation frequency over controls under BL activation at a concentration of 5 micrograms/ml; marmesin induced a 2-fold increased mutation frequency at 1.5 micrograms/ml; MOP induced a 19-fold increase at 10 micrograms/ml; chalepin induced a 3-fold increase at 5 micrograms/ml; and imperatorin induced a 20-fold increase at 10 micrograms/ml. Essentially no mutation was observed at the ouabain-resistant (Ouar) locus in C3H/1OT1/2 cells with any of these compounds. In the transformation assays, type II and type III foci were observed at a 1-microgram/ml addition of AFB1 with or without BL activation; while with MOP and imperatorin, these types of foci were observed only with BL activation. Marmesin, although relatively more cytotoxic than the other furocoumarins studied, with a 50% lethal dose of less than 0.5 micrograms/ml, was not as mutagenic or potentially carcinogenic as were AFB1, imperatorin, or MOP with BL activation. These furocoumarins are considered to be involved in the etiology of the high incidence of skin cancer in Nigeria. Our experiments reinforce that concept and suggest that exposure to these furocoumarins may constitute a real carcinogenic hazard.     

Glutathione S-transferase localization in aflatoxin B1-treated rat livers

Overexpression of detoxication enzymes is associated with the development of drug-resistant, preneoplastic nodules in the carcinogen-treated rat liver. The most consistent marker of preneoplasia in many experimental models is increased expression of the pi-class glutathione S-transferase (GST) YfYf. We have confirmed by immunostaining that the pi-class GST is overexpressed in aflatoxin B1-induced preneoplastic nodules and liver tumours in rats. However, pi-class GST YfYf has low activity against aflatoxin B1-8,9-epoxide, and most activity against this cytotoxic and genotoxic metabolite is associated with the alpha-class GSTs YaYa, YaYc and YcYc. We have demonstrated that there is also a consistent increase in the alpha-class GSTs in this model. It seems likely that the overexpression of the Ya and Yc subunits, rather than increased levels of the pi-class GST YfYf, is responsible for the acquisition of a drug-resistant phenotype in rat liver preneoplastic nodules and tumours induced by aflatoxin B1.     

Changes in adult metabolism of aflatoxin B1 in rats neonatally exposed to diethylstilbestrol. Alterations in alpha-class glutathione S-transferases.

Neonatal exposure of rats to xenobiotics has been shown to produce long-term alterations in hepatic enzyme activities and in levels of DNA adducts following carcinogen exposure. We exposed newborn male rats to diethylstilbestrol (DES), pregnenolone-16 alpha-carbonitrile, 7,12-dimethylbenz[a]anthracene or phenobarbital on days 1, 3 and 5 of age. At five months of age, males were injected with 1 mg/kg of [3H]aflatoxin B1 (AFB1), killed after 2 h and examined for AF-DNA adduction in the liver. Males neonatally exposed to DES showed a 35% decrease in DNA adduction levels. Analysis of the adducted DNA bases failed to show any changes in relative proportions of individual adducts in the DES samples compared to controls. Hepatic glutathione concentrations were unchanged. However, Western blot analysis of alpha-class glutathione S-transferases (alpha GST), enzymes known to inactivate the toxic AFB1-8,9-epoxide, showed a 2-fold increase in subunit levels in the DES-treated males, suggesting that the detoxifying activity of the cytosol may have been increased. To confirm this, in vitro tests were undertaken using butylated hydroxyanisole (BHA) induced mouse microsomes to activate [3H]AFB1 in the presence of treated cytosol and GSH. Analysis of metabolites by HPLC showed that DES-treated males formed 245% of the AFB-SG conjugate relative to vehicle controls. These results indicate that neonatal DES treatment resulted in long-term changes in basal alpha GST levels and suggest that these changes were responsible for lower levels of DNA adduction following adult exposure to AFB1.     

Specificity of murine glutathione S-transferase isozymes in the glutathione conjugation of (-)-anti- and (+)-syn-stereoisomers of benzo[g]chrysene 11,12-diol 13,14-epoxide.

Specificities of murine glutathione (GSH) S-transferase (GST) isozymes mGSTA1-1, mGSTA2-2, mGSTA3-3 and mGSTA4-4 (alpha class), mGSTP1-1 (pi class) and mGSTM1-1 (mu class) for GSH conjugation of (-)-anti- and (+)-syn-stereoisomers of benzo[g]chrysene 11, 12-diol 13,14-epoxide (B[g]CDE), the activated metabolites of the environmental pollutant benzo[g]chrysene (B[g]C), have been determined. When GST activity was determined as a function of varying (-)-anti- or (+)-syn-B[g]CDE concentration (10-320 microM) at a fixed saturating concentration of GSH (2 mM), each isozyme obeyed Michaelis-Menten kinetics. mGSTA1-1 was significantly more efficient than other murine GSTs in the GSH conjugation of not only (-)-anti-stereoisomer but also (+)-syn-B[g]CDE. For example, the catalytic efficiency (k(cat)/K(m)) of mGSTA1-1 towards (-)-anti-B[g]CDE was approximately 2.3- to 16.6-fold higher compared with other murine GSTs. Likewise, mGSTA1-1 was approximately 2.7-, 6.7-, 4.4- and 12.4-fold more efficient than mGSTA2-2, mGSTA3-3, mGSTP1-1 and mGSTM1-1, respectively, in catalyzing the GSH conjugation of (+)-syn-B[g]CDE. Interestingly, mGSTA4-4, which also belongs to class alpha, was virtually inactive towards both stereoisomers of B[g]CDE. The results of the present study indicate that murine GSTs, especially alpha class isozymes, significantly differ in their ability to detoxify B[g]CDE stereoisomers and that mGSTA1-1 plays a major role in the detoxification of both (-)-anti- and (+)-syn-B[g]CDE, which among four B[g]CDE stereoisomers are formed from the carcinogen B[g]C as major DNA binding metabolites.     

B7-1基因转染骨肉瘤细胞诱导抗骨肉瘤主动免疫的实验研究

目的:探讨B7-1基因转染骨肉瘤细胞能否诱导抗骨肉瘤主动免疫作用。方法:利用脂质体将B7-1真核表达载体pcDNA3-B7-1和空载体pcDNA3分别导入骨肉瘤细胞株LM8中,G418筛选出阳性克隆(分别命名为LM8/B7-1和LM8/pcDNA3),通过RT-PCR、Western blot和流式细胞仪检测B7-1基因与蛋白的表达;通过软琼脂克隆形成实验观察转基因细胞株LM8/B7-1的体外增殖能力;用LM8、LM8/B7-1和LM8/pcDNA3分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞和致敏脾细胞,MTT法检测其体外杀伤活力;将LM8、LM8/B7-1和LM8/pcDNA3细胞分别接种到C3H雄性小鼠前腋下,观察其致瘤能力;观察LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8是否具有免疫保护作用。结果:B7-1基因在转基因细胞LM8/B7-1中能得到高表达;LM8/B7-1体外增殖能力与LM8和LM8/pcDNA3无明显差异(P>0.05);LM8/B7-1诱导的CTL的杀伤活力显著高于LM8和LM8/pcDNA3诱导的CTL对相同靶细胞的杀伤活力,LM8/B7-1诱导的CTL对LM8/B7-1的杀伤活力也明显高于对LM8和LM8/pcDNA3的杀伤活力(P<0.05);LM8/B7-1致瘤能力较LM8和LM8/pcDNA3细胞明显下降(P<0.01);LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8具有免疫保护作用。结论:B7-1基因转染骨肉瘤细胞能诱导抗骨肉瘤主动免疫作用,为利用B7-1基因进行骨肉瘤免疫基因治疗提供了实验依据。     

Mechanisms of protection against aflatoxin B(1) genotoxicity in rats treated by organosulfur compounds from garlic

Diallyl sulfide (DAS) and diallyl disulfide (DADS), two garlic constituents, were found previously to inhibit aflatoxin B(1) (AFB(1))-initiated carcinogenesis in rat liver, DADS being the most effective. In order to study the mechanisms involved in this protection, we have examined the ability of liver microsomes and cytosols from DAS- and DADS-treated rats to modulate the mutagenicity and the metabolism of AFB(1). We also examined the effects of these compounds on the expression of cytochromes P450 (CYP) and phase II enzymes known to be involved in AFB(1) metabolism. Administration of DAS (1 mmol/kg for 4 days) to rats resulted in significant inhibition of microsome-mediated mutagenicity of AFB(1), whereas DADS treatment did not alter AFB(1) mutagenicity. DAS treatment increased the metabolism of AFB(1) mainly towards the formation of AFQ(1) and AFM(1), which might account for the reduction of AFB(1) microsomal-mediated mutagenicity. DADS treatment slightly affected the oxidative metabolism of AFB(1). DAS and DADS induced CYP3A2, CYP2B1 and CYP2B2, DAS being more potent. Cytosols from DAS- and DADS-treated rats produced a significant inhibition of AFB(1)-8,9-epoxide (AFBO)-induced mutagenicity and significantly increased the cytosolic formation of AFB(1)-glutathione conjugates, DADS treatment being more effective. Western blot analysis showed that DADS is a potent inducer of glutathione S-transferase A5 (rGSTA5) and AFB(1) aldehyde reductase 1 (rAFAR1), while DAS is a weak inducer of these enzymes. Finally, we demonstrated that antibodies raised against rGSTA5 strongly reduced the antimutagenic activity of cytosols from DAS- and DADS-treated rats against AFBO. All together, these results demonstrate that DAS prevents AFB(1) mutagenicity through a dual mechanism, i.e. by modulating both the phase I and II metabolism of AFB(1), whereas DADS acts mainly by increasing the phase II metabolism of AFB(1). The induction of rGSTA5 and rAFAR1 is probably the main mechanism by which allyl sulfides give protection against AFB(1)-induced carcinogenesis.     

Potent protective effect of isoimperatorin against aflatoxin B1-inducible cytotoxicity in H4IIE cells: bifunctional effects on glutathione S-transferase and CYP1A

Typically chemopreventive agents either induce phase II detoxifying enzymes or inhibit the cytochrome P450 enzymes (CYPs) that are required for the metabolism of carcinogens. In this study, we isolated a coumarin compound, isoimperatorin from Poncirus trifoliata Raf., and studied its protective effects against aflatoxin B1 (AFB1)-induced cytotoxicity in H4IIE cells. Isoimperatorin (>0.3 microM) significantly inhibited the cytotoxic effect of AFB1. CDNB [1-chloro-2,4-dinitrobenzene; glutathine S-transferase (GST) subtype-non-specific] and NBD (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; GSTalpha type-specific) assays revealed that isoimperatorin (0.3-3 microM) increased GST activity in a concentration-dependent manner. Western blot analyses using subtype-specific antibodies confirmed that GSTalpha protein, but not GSTmu or GSTpi, was induced in cells treated with isoimperatorin. Reporter gene analysis using an antioxidant response element (ARE) containing construct and subcellular fractionation assays revealed that GSTalpha induction by isoimperatorin is associated with Nrf2/ARE activation. Moreover, ethoxyresorufin-O-deethylase assays showed that isoimperatorin (2 microM) completely inhibited 3-methylchoranthrene-inducible CYP1A activity. These results indicate that isoimperatorin from Poncirus trifoliata Raf. possesses a potent hepatoprotective effect against AFB1, presumably through the induction of GSTalpha and the direct inhibition of CYP1A, and suggest that isoimperatorin should be considered a potential chemopreventive.