Protective effect of soybean saponins and major antioxidants against aflatoxin B1-induced mutagenicity and DNA-adduct formation

Saponins from various plant sources have been suggested as possible anticarcinogens. Major dietary sources of saponins include legumes such as soybeans. This study was performed to determine the effect of soybean saponins on aflatoxin B(1)(AFB(1))-induced mutagenicity and AFB(1)-DNA adduct formation using Salmonella typhimurium and human liver hepatoma (HepG2) cells, respectively. Major antioxidants including L-ascorbic acid, alpha-tocopherol, all-trans-retinol, and butylated hydroxytoluene (BHT), previously reported to possess antimutagenic activity, were used as test materials to evaluate the relative effectiveness of saponins. Results indicated antimutagenicity was in the order of BHT > saponins > alpha-tocopherol > L-ascorbic acid. Soybean saponins exerted a significant effect, inhibiting the mutagenicity of AFB(1) by 52%, 64%, and 81% at concentrations of 600, 900, and 1,200 microg per plate, respectively. The amount of tritiated AFB(1) metabolites-DNA adducts formed in HepG2 cells was significantly reduced when cells were preincubated with 10 or 30 microg/ml of test materials. Soybean saponins inhibited AFB(1)-DNA adduct formation by 50.1% at a concentration of 30 microg/ml, whereas L-ascorbic acid and BHT reduced adduct formation by 38.4% and 32.6%, respectively, at the same concentrations. These results indicate that soybean saponins possess not only a significant antimutagenic activity but a strong inhibitory action against carcinogen-induced DNA damages. Soybean saponins possibly block the initiation stage of carcinogenesis, and further studies are required to elucidate the mechanisms of action.     

Aflatoxin B1 DNA-Adducts in Hepatocellular Carcinoma from a Low Exposure Area

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell populations displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas.     

镰刀菌毒素与黄曲霉毒素的联合毒性研究--AFB1和DON的RDS实验

为了解黄曲霉毒素（ＡＦＢ１）与镰刀菌毒素的脱氧雪腐镰刀菌烯醇（ＤＯＮ）对ＤＮＡ损伤修复的影响,用化学物质诱导的细胞增殖的变化实验,（ｒｅｐｌｉｃａｔｉｖｅ ＤＮＡ ｓｙｎｔｈｅｓｉｓ,ＲＤＳ）实验进行研究。结果表明：ＡＦＢ１和ＤＯＮ均可诱导细胞分化的Ｓ期ＤＡＮ的合成,并且二者间存在交互作用。结果提示：ＡＦＢ１与ＤＯＮ在肿瘤的发生中既以遗传毒性物质的形式发挥作用,又以非遗传毒性物质的形式共同发挥作用     

Effects of dietary selenium and vitamin E on covalent binding of aflatoxin to chick liver cell macromolecules

Day-old single comb white Leghorn chicks of both sexes maternally depleted in selenium (Se) and vitamin E (VE) were fed a low Se and VE-free semipurified basal diet or that diet supplemented with graded levels of Se (0.2 - 20.0 ppm as Na2SeO3) of VE (100 IU/g as all-rac-alpha-tocopheryl acetate), or both. At 14 days of age, chicks were given 1 mg/kg [3H] aflatoxin B1 (AFB1) i.p. and killed either 2 or 24 hours later. Covalent binding of AFB1 to liver DNA and RNA in chicks fed the basal diet was significantly greater than in chicks supplemented with Se or VE, or both. Phenobarbital treatment prior to administration of AFB1 decreased adduct formation in most groups, and abolished differences in adduct formation due to diet. These results suggest that combined Se-VE deficiency enhances activation or inhibits detoxification of AFB1 in vivo.     

Organosulfur compounds of garlic modulate mutagenesis, metabolism, and DNA binding of aflatoxin B1

The effects of two organosulfur compounds of garlic (ajoene and diallyl sulfide) and a crude garlic extract on aflatoxin B1 (AFB1)-induced mutagenesis were determined using rat liver 9,000 g supernatant (S-9) as the activation system and Salmonella typhimurium TA-100 as the tester strain. The effects of these compounds on AFB1 binding to calf thymus DNA were also measured. Metabolites of AFB1 were isolated and analyzed by reverse-phase high-performance liquid chromatography. All these compounds inhibited S-9-dependent mutagenesis induced by AFB1. They also inhibited AFB1 binding to DNA. A significant decrease in organo-soluble metabolites of AFB1 was observed with ajoene and garlic extract. An increase of glucuronide and glutathione conjugates was obtained with garlic extract. The results indicate that garlic compounds tested in this study are antimutagenic and, potentially, anticarcinogenic.     

Associations of plasma aflatoxin B1-albumin adduct level with plasma selenium level and genetic polymorphisms of glutathione S-transferase M1 and T1

Mortality from hepatocellular carcinoma (HCC) is extraordinarily high in Matzu, an island off the coast of Southeastern China. To investigate factors associated with plasma aflatoxin B1 (AFB1)-albumin adduct level, we studied 304 healthy adult residents from Matzu. AFB1-albumin adducts were determined by competitive enzyme-linked immunosorbent assay, hepatitis B surface antigen status by enzyme immunoassay, genotypes of glutathione S-transferase (GST) M1 and T1 by polymerase chain reaction, plasma selenium by atomic absorption spectrometry, and plasma retinol, alpha-tocopherol, alpha-carotene, and beta-carotene levels by high-performance liquid chromatography. Men had higher AFB1-albumin adduct levels than women. GSTM1-nonnull and GSTT1-null genotypes and low plasma selenium level were significantly associated with an increased level of AFB1-albumin adducts among men, whereas age was significantly correlated with adduct level among women. High intake of fermented beans was associated with an increased adduct level among men and women. The inverse associations between plasma selenium level and AFB1-albumin adducts were statistically significant among those with null genotypes of GSTM1 and GSTT1, but not among the nonnull genotypes. This study provides insight into the dietary and genetic factors influencing AFB1-albumin adduct formation in an isolated population with high liver cancer mortality.     

Effects of capsaicin on rat liver S9-mediated metabolism and DNA binding of aflatoxin

At concentrations of 25, 50, and 100 microM, capsaicin, which is the major component in various aspects of Capsicum hot peppers, decreased the binding of aflatoxin (AFB1) to calf thymus DNA by 19%, 44%, and 71%, respectively, in incubations with rat liver S9. At concentrations of 50 and 100 microM, capsaicin decreased the formation of AFB-DNA adducts (AFB1-N7-Gua) by 53% and 75% as determined by high-pressure liquid chromatography (HPLC). HPLC analysis of organo-soluble fractions showed that these effects correlated with a concentration-dependent decrease in S9-mediated metabolism of AFB1 by capsaicin. Capsaicin also altered the formation of water-soluble conjugates of AFB1. This was indicated by a decrease in radioactivity in water-soluble fractions and in glutathione conjugates of AFB1 analyzed by HPLC. These results suggest that capsaicin inhibited the biotransformation of AFB1 by modifying Phase I hepatic enzyme activity.     

Effect of carnitine on propionate metabolism in the vitamin B-12--deficient rat

Acyl-CoA thioesters are generated during the oxidation of organic acids in mammalian systems. Vitamin B-12 deficiency is associated with decreased L-methylmalonyl-CoA mutase activity, and consequent accumulation of propionyl-CoA and methylmalonyl-CoA. The formation of propionylcarnitine from propionyl-CoA and carnitine provides an alternative pathway to remove propionyl-CoA from cells. Hepatocytes isolated from vitamin B-12--deficient rats metabolized propionate (1 mM) to CO2 and glucose at only 23% and 12%, respectively, of the rates observed in hepatocytes from control animals. In contrast, no difference was seen in rates of pyruvate metabolism by hepatocytes from control and vitamin B-12--deficient rats. Addition of carnitine (10 mM) to hepatocyte incubations increased the rate of propionylcarnitine formation 10- to 20-fold without altering conversion of propionate to CO2 or glucose. The rate of propionylcarnitine formation was not affected by vitamin B-12 deficiency. When carnitine (10 mM) was added, propionylcarnitine generation represented 65-71% of total propionate utilization in hepatocytes isolated from vitamin B-12--deficient rats. Gluconeogenesis from [1-14C]pyruvate was inhibited by 1 mM propionate in hepatocytes from vitamin B-12--deficient rats. No effect of 1 mM propionate on glucose formation from pyruvate was seen using hepatocytes from control rats. Intraperitoneal administration of L-carnitine resulted in a significant increase in urinary propionylcarnitine excretion from vitamin B-12--deficient rats, but not from control animals. The results demonstrate that exogenous carnitine can significantly enhance propionyl-group utilization via the formation of acylcarnitines under the conditions of impaired acyl-CoA metabolism associated with vitamin B-12 deficiency.     

穿梭质粒pSP189/哺乳动物细胞系统在AFB_1诱变分子机理研究中的应用

ＡｆｌａｔｏｘｉｎＢ１（ＡＦＢ１）是食品中广泛存在的致癌性最强的霉菌毒素之一，本文将基于ＳＶ４０病毒的短暂复制型穿梭质粒ｐＳＰ１８９与非洲绿猴肾细胞（ＶｅｒｏＥ６细胞系）组成穿梭质粒／哺乳动物细胞诱变检测系统检测ＡＦＢ１的诱变性，并通过对靶基因ＳｕｐＦＴＲＮＡ的序列分析了解ＡＦＢ１在ＤＮＡ一级结构上的作用位点，类型及序列特异性等信息。结果表明：在体外采用大鼠肝微粒体与ＡＦＢ１和ｐＳＰ１８９直接作用形成ＡＦＢ１－ＤＮＡ加合物，再转染ＶｅｒｏＥ６细胞，随着ＡＦＢ１作用质粒ＤＮＡ时间的延长，经哺乳动物细胞内复制并在宿主菌中筛选得到的突变体逐渐增加，并呈明显的剂量－反应关系，检出的突变体经０．８％琼脂糖凝胶电泳分析大多为点突变，对其中５３个独立突变子的靶基因ＳｕｐＦＴＲＮＡ的序列分析结果表明：ＡＦＢ１诱发ｐＳＰ１８９靶基因ＳｕｐＴＩＲＮＡ的突变大多单碱基置换，占突变子总数的８４．９％，９５．２％的碱基置换发生在ＧＣ位点，其中以ＧＣ→ＴＡ的颠换占大多数，约为５３．３％，其次为ＧＣ→ＡＴ的转换，约为３５．６％；ＡＦＢ１诱发的突变在靶基因ＳｕｐＦＴＲＮＡ的分布并非是随机的，而是存在着一定的序列特异性，其特征序列为“５＇－?     

Acetylcarnitine-mediated inhibition of ethanol oxidation in hepatocytes

Carnitine-mediated prevention of ethanol-induced hepatic steatosis is related to the attenuation of ethanol metabolism by carnitine in the intact rat. Although carnitine retards ethanol oxidation in the intact animal, the in vitro activities of ethainol-metabolizing enzymes remain unaltered. Therefore, hepatocytes were targeted to understand the mechanism of carnitine effect on ethanol metabolism. Rat hepatocytes were isolated by a collagenase-perfusion technique and incubated in albumin-containing medium with ethanol in the presence or absence of added carnitine or related compounds. Ethanol oxidation was determined by the loss of ethanol as well as by the products formed. The rate of ethanol oxidation in the presence of carnitine was one-half the rate in the absence of carnitine (14 vs. 25 nmol · min⁻¹ · million⁻¹ cells). It took 100 times the concentration of carnitine to equal the maximal inhibition produced by acetylcarnitine and the effect of acetylcanitine was without a lag time. It is concluded that acetylcarnitine is the mediator of carnitine inhibition of ethanol oxidation.     

Variability in aflatoxin-albumin adduct levels and effects of hepatitis B and C virus infection and glutathione S-transferase M1 and T1 genotype

Exposure to aflatoxin B1 (AFB1), an important cofactor in the etiology of hepatocellular carcinoma in Taiwan, is influenced by dietary and other factors. The present study examined the intraindividual variability in AFB1-albumin adducts, the most reliable long-term biomarker of AFB1 exposure, and whether the baseline or follow-up adduct levels and the intraindividual variability in adduct levels are modified by endogenous and environmental factors. The study measured AFB1-albumin adduct levels among 264 healthy male residents of three townships (Hu-Hsi, Ma-Kung, and Pai-Hsa) of Penghu Islets, Taiwan, at two different time points with a median interval of 1.68 years (range 1.00-3.17 years). There was a generalized reduction in the adduct levels, with the median values being 22.1 pmol/mg (range 5.0-355.8 pmol/mg) at time 1 and 14.3 pmol/mg (range 5.0-205.2 pmol/mg) at time 2. This intraindividual variability in adduct levels was inversely associated with the age of subjects and the time interval between the two blood draws. The variability in adduct levels was lower among subjects in Hu-Hsi and Pai-Hsa townships as compared to those in Ma-Kung. No significant association was observed for the intraindividual variability in AFB1-albumin adducts with regard to the season when blood was drawn. There was also no significant association between intraindividual variability and hepatitis B surface antigen, anti-hepatitis C virus (anti-HCV), glutathione S-transferase (GST) M1, or GSTT1 status. In conclusion, we found substantial intraindividual variability in the AFB1 exposure (as determined by AFB1-albumin adducts) in Taiwan, which was probably more likely related to dietary or other environmental influences rather than to endogenous factors (e.g., hepatitis B/C viral infection or GST M1/T1 genetic status).     

Carnitine import to isolated hepatocytes and synthesis are accelerated in pivalate-treated rats.

To investigate the effect of pivalate on carnitine import and carnitine synthesis in the liver, we measured carnitine uptake in isolated rat hepatocytes with L-[(14)C] carnitine and concentrations of free carnitine, gamma-butyrobetaine and acylcarnitines using tandem mass spectrometry. Hepatocytes from rats treated with 20 mmol/L of pivalate for 4 wk had greater L-[(14)C] carnitine uptake than those of unsupplemented rats after 5, 10, 30 and 90 min. Addition of 1 mmol/L of pivalate or 1 mmol/L of pivaloylcarnitine to control cell suspensions did not affect L-[(14)C] carnitine uptake. The K(m) values for L-[(14)C] carnitine uptake for pivalate-treated rats were significantly lower than control (2.9 +/- 0.7 mmol/L for pivalate-treated rats, 6.2 +/- 1.1 mmol/L for controls). The concentration of free carnitine was not reduced in the liver of pivalate-treated rats, whereas the concentrations of acetylcarnitine and gamma-butyrobetaine were significantly lower than controls. In the heart and muscle the concentration of free carnitine was significantly lower and that of gamma-butyrobetaine was higher than controls. These results suggest that carnitine transport from plasma into the liver and synthesis in the liver are accelerated in rats with secondary carnitine deficiency induced by the administration of pivalate.     

Associations of serum aflatoxin B1-lysine adduct level with socio-demographic factors and aflatoxins intake from nuts and related nut products in Malaysia

Aflatoxins are one of the major risk factors in the multi-factorial etiology of human hepatocellular carcinoma. Therefore, the information on aflatoxins exposure is very important in the intervention planning in order to reduce the dietary intake of aflatoxins, especially among the children. This study investigated the relationship between aflatoxin B(1) (AFB(1)) lysine adduct levers in serum and socio-demographic factors and dietary intake of aflatoxins from nuts and nut products in Penang, Malaysia. A cross-sectional field study was conducted in five districts of Penang. A survey on socio-demographic characteristics was administered to 364 healthy adults from the three main ethnic groups (Malay, Chinese and Indian). A total of 170 blood samples were successfully collected and tested for the level of AFB(1)-lysine adduct. 97% of the samples contained AFB(1)-lysine adduct above the detection limit of 0.4 pg/mg albumin and ranged from 0.20 to 23.16 pg/mg albumin (mean±standard deviation=7.67±4.54 pg/mg albumin; median=7.12 pg/mg albumin). There was no significant association between AFB(1)-lysine adduct levels with gender, district, education level, household number and occupation when these socio-demographic characteristics were examined according to high or low levels of AFB(1)-lysine. However, participants in the age group of 31-50 years were 3.08 times more likely to have high AFB(1) levels compared to those aged between 18 and 30 years (P=0.026). Significant difference (P=0.000) was found among different ethnic groups. Chinese and Indian participants were 3.05 and 2.35 times more likely to have high AFB(1) levels than Malay. The result of AFB(1)-lysine adduct suggested that Penang adult population is likely to be exposed to AFB(1) but at a level of less than that needed to cause direct acute illness or death.     

Tea as a potential chemopreventive agent in PhIP carcinogenesis: effects of green tea and black tea on PhIP-DNA adduct formation in female F-344 rats

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of proteinaceous animal foods (meat, chicken, and fish). PhIP is a carcinogen in the Fischer 344 (F-344) rat; it induces mammary tumors in female rats and lymphomas and colon and prostate tumors in male rats. In F-344 rats, PhIP forms DNA adducts in various organs, including the target organs. Inhibition of PhIP-DNA adduct formation is likely to lead to inhibition of PhIP tumorigenicity. We have examined the chemopreventive properties of green tea and black tea in PhIP carcinogenesis by evaluating their effects on PhIP-DNA adduct formation in the female F-344 rat. Young adult animals were maintained on powdered AIN-76A diet while receiving regular drinking water or 2% (wt/vol) infusions of green tea or black tea for a total of six weeks. During Weeks 3, 4, and 5, all animals received PhIP by gavage (1 mg/kg/day). Three rats per group were euthanized on Days 1 and 8 after termination of PhIP exposure. DNA was isolated from a number of organs and analyzed for PhIP-DNA adducts by 32P-postlabeling assays. Compared with animals on regular drinking water, PhIP-DNA adduct formation was inhibited in small intestine, colon, liver, and mammary epithelial cells (MECs) of animals receiving green tea or black tea as the sole source of drinking fluid. Green tea inhibited adduct formation in colon, liver, and MECs (33.3-80.0%) on both days, but only on Day 8 (54.4%) in small intestine. Black tea inhibited adduct formation on both days in liver (71.4-80.0%), on Day 1 in colon (40.0%), and on Day 8 in small intestine (81.8%); it had no effect on MEC adducts. Neither green tea nor black tea had an effect on adduct levels in pancreas, lungs, white blood cells, heart, kidneys, spleen, cecum, or stomach. Similarly, these teas did not affect the rate of adduct removal (percent change from Day 1 to Day 8) in any organ. It is concluded that green tea and black tea are potential chemopreventive agents in PhIP-induced tumorigenesis in the F-344 rat.     

抗黄曲霉毒素B1单抗制备及免疫学定量方法建立

目的本研究合成并鉴定了AFB1人工抗原,制备AFB1的单克隆抗体(AFB1mAb)。方法采用NHS法将AFB1分别偶联于载体蛋白BSA和OVA上,分别合成W人工抗原AFB1-BSA和AFB1-OVA,紫外分光光度法和SDS-PAGE进行鉴定;AFB1-BSA免疫BALB/C小鼠,通过间接ELISA和阻断ELISA法选择细胞融合备用鼠;用杂交瘤技术制备AFB1mAb,并对其效价、亲和力、敏感性、特异性、亚型进行鉴定;体内诱生腹水法大量制备单抗。结果 UV图谱和SDS-PAGE图表明结半抗原AFB1和载体BSA及OVA偶联成功;筛选出2H5-F6、2H5-C9、2H9-C3三株杂交瘤细胞;鉴定单抗亚型均为IgG1;细胞上清效价1∶2.0×102～1∶1.28×103,2H5-F6的腹水效价1∶1.28×106,AFB1mAb亲和常数Ka为2.65×1010 L/moL,对AFB1的IC50为2.58 ng/mL;与AFB2的交叉反应率为1.61%,与其他类药物无交叉反应。结论通过试验获得高效价、敏感、特异的AFB1mAb,可用于各种食品中AFB1残留的快速免疫学检测试验。     

黄曲霉毒素B1诱导性大鼠肝癌超微结构观察及N-ras表达变化

目的探讨黄曲霉毒素B1(AFB1)诱导性大鼠肝癌模型超微病理特征及N-rasmRNA表达变化规律。方法 40只Wistar大鼠分为正常对照组(12只)和诱癌组(28只)用AFB1(400μg/kg)间断腹腔注射雄性Wistar大鼠制作肝癌模型,电镜观察大鼠肝组织超微结构。应用RT-PCR技术检测对照组大鼠肝组织,损伤病变、早期癌变和癌变肝组织中N-ras mRNA表达水平。结果本组大鼠肝癌模型中,肝细胞呈变性损伤,异型性到癌变;线粒体由增生肿胀到枯竭、空泡变;糖原颗粒呈逐渐减少等特征性变化。观察到典型肝细胞吞噬细胞现象。N-rasmRNA在早期癌变组织、癌变组织中表达水平均显著高于对照组大鼠肝组织和损伤病变肝组织(F=5.47,P=0.019;后者F=6.98,P=0.006)。结论 AFB1诱导性大鼠肝细胞出现变性、异型性及癌细胞渐变的超微结构特征,N-ras异常表达参与大鼠肝细胞癌变机制。     

Interaction of carnitine and propionate with pyruvate oxidation by hepatocytes from clofibrate-treated rats: importance of coenzyme A availability.

Propionate interferes with normal hepatic metabolic regulation secondary to accumulation of propionyl- and methylmalonyl-CoA. Clofibrate-treatment increases hepatic CoA content and carnitine acetyltransferase activity, both of which may modulate propionate toxicity. Therefore, inhibition of pyruvate oxidation by propionate was studied in hepatocytes isolated from rats maintained on a control or 0.5% clofibrate diet for 7-9 d. Propionate (10 mmol/L) inhibited 14CO2 formation from [1-14C]pyruvate (10 mmol/L) by 60 +/- 2% in hepatocytes from control rats, but by only 46 +/- 3% in cells from clofibrate-treated rats (P less than 0.05). The smaller inhibitory effect of propionate in hepatocytes from clofibrate-treated rats occurred despite increased cellular propionyl-CoA content as compared with controls, but was associated with increased CoASH and total CoA contents. Despite greater carnitine acetyltransferase activity (20-fold) and propionylcarnitine production (2.5-fold) in hepatocytes from clofibrate-treated rats, reversal of propionate's inhibition of pyruvate oxidation by 10 mmol/L carnitine was small (8.7 +/- 3.9%) and not different from that observed in cells from control animals (6.7 +/- 2.4%). Carnitine (10 mmol/L) decreased hepatocyte total acid-soluble CoA content by 20-30% in cells from both control and clofibrate-treated rats. This carnitine-induced decrease in CoA content may limit the efficacy of carnitine under conditions of acyl-CoA accumulation. Clofibrate-induced increased CoA content provides partial protection against propionate toxicity. Metabolic toxicity of propionate is the result of both the increased cellular propionyl-CoA content and the depletion of cellular unesterified CoA.     

Inhibition of DNA adduct formation of PhIP in female F344 rats by dietary conjugated linoleic acid

The dietary mutagen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is a mammary carcinogen in the female Fischer (F344) rat and a colon carcinogen in the male F344 rat. To exert its carcinogenicity, it is believed that PhIP needs to form adducts with DNA, a process requiring N‐hydroxylation of PhIP by cytochromes P‐450 1A1 and/or 1A2 (CYP 1A1 and/or 1A2), as well as further esterification of the hydroxylamine thus formed. Dietary conjugated linoleic acid (CIA) inhibits chemical carcinogenesis in various experimental models. We have examined the effect of dietary CLA on PhIP‐DNA adduct formation in female F344 rats. Four‐week‐old animals were maintained on AIN‐76A diet without or with CLA (1%, 0.5%, and 0.1% wt/wt) for 57 days. PhIP was added to the diets (0.04% wt/wt) from Days 14–42. Animals were killed (4/group) on Days 43, 50, and 57. DNA isolated from liver, mammary epithelial cells (MEC), colon, and white blood cells (WBC) was analyzed for PhIP‐DNA adducts by ³²P‐postlabeling assays. On Day 43, CIA inhibited adduct formation in the liver (up to 58%) in a dose‐dependent manner. CIA also inhibited hepatic adduct levels (29–39%) on Day 50 (at 1.0% and 0.5% CIA) and on Day 57 (53% at 0.5% CLA). CLA significantly reduced adduct levels in the WBC on Day 50 (63–70%). Adducts in MEC and the colon were not affected by dietary CIA. On Day 57, adduct levels in MEC, liver, colon, and WBC were 0–30.3%, 8.6–41.7%, 21.5–50.7%, and 7.5–11.8%, respectively, of those on Day 43. Northern blot analysis of liver RNA showed that dietary CIA did not affect steady‐state levels of CYP 1A1 or 1A2 mRNA. It is concluded that dietary CLA inhibits PhIP‐DNA adduct formation in liver and WBC but that those in MEC and the colon are unaffected when a low‐level dietary regimen of carcinogen and inhibitor was used. In inhibiting PhIP‐DNA adduct formation, CIA does not appear to act by inhibiting CYP 1A1 or 1A2 expression.     

Dual effect of ethanol on cell death in primary culture of human and rat hepatocytes

AIMS: In-vivo and in-vitro studies have shown that ethanol induces hepatocyte damage. The aim of the present study was to evaluate the effect of a broad range of ethanol concentrations on apoptosis and necrosis in primary culture of human and rat hepatocytes. METHODS: Human and rat hepatocytes were isolated from human hepatectomies and male Wistar rats (200-250 g) using the classical collagenase perfusion method. After stabilization of cell culture, ethanol (0-10 mmol/l) was administered and the parameters were measured 24 h after ethanol addition. Apoptosis was studied by DNA fragmentation, iodide propidium-DNA staining, caspase-3 activity and annexin V binding in hepatocytes. Necrosis was evaluated by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) and GSH/GSSG were used as parameters of oxidative stress. RESULTS: Ethanol enhanced dose-dependently all the parameters associated with apoptosis in human and rat hepatocytes. Low or high ethanol concentrations induced an opposite action against cell necrosis in cultured hepatocytes. Low concentrations of ethanol (1-2 mmol/l) reduced LDH release from human and rat hepatocytes. However, the highest ethanol concentration (10 mmol/l) induced a sharp increase in cell necrosis. The effect of ethanol on cell necrosis was related to lipid peroxidation in hepatocytes. CONCLUSIONS: Ethanol differentially regulates apoptosis or necrosis in cultured hepatocytes. Although ethanol exerted a dose-dependent induction of apoptosis, low ethanol concentrations were able to reduce basal lipid peroxidation and necrosis in hepatocytes. The highest ethanol concentration (10 mmol/l) induced apoptosis and necrosis in human and rat cultured hepatocytes.     

Inhibition of DNA adduct formation of PhIP in female F344 rats by dietary conjugated linoleic acid

The dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary carcinogen in the female Fischer (F344) rat and a colon carcinogen in the male F344 rat. To exert its carcinogenicity, it is believed that PhIP needs to form adducts with DNA, a process requiring N-hydroxylation of PhIP by cytochromes P-450 1A1 and/or 1A2 (CYP 1A1 and/or 1A2), as well as further esterification of the hydroxylamine thus formed. Dietary conjugated linoleic acid (CLA) inhibits chemical carcinogenesis in various experimental models. We have examined the effect of dietary CLA on PhIP-DNA adduct formation in female F344 rats. Four-week-old animals were maintained on AIN-76A diet without or with CLA (1%, 0.5%, and 0.1% wt/wt) for 57 days. PhIP was added to the diets (0.04% wt/wt) from Days 14-42. Animals were killed (4/group) on Days 43, 50, and 57. DNA isolated from liver, mammary epithelial cells (MEC), colon, and white blood cells (WBC) was analyzed for PhIP-DNA adducts by 32P-postlabeling assays. On Day 43, CLA inhibited adduct formation in the liver (up to 58%) in a dose-dependent manner. CLA also inhibited hepatic adduct levels (29-39%) on Day 50 (at 1.0% and 0.5% CLA) and on Day 57 (53% at 0.5% CLA). CLA significantly reduced adduct levels in the WBC on Day 50 (63-70%). Adducts in MEC and the colon were not affected by dietary CLA. On Day 57, adduct levels in MEC, liver, colon, and WBC were 0-30.3%, 8.6-41.7%, 21.5-50.7%, and 7.5-11.8%, respectively, of those on Day 43. Northern blot analysis of liver RNA showed that dietary CLA did not affect steady-state levels of CYP 1A1 or 1A2 mRNA. It is concluded that dietary CLA inhibits PhIP-DNA adduct formation in liver and WBC but that those in MEC and the colon are unaffected when a low-level dietary regimen of carcinogen and inhibitor was used. In inhibiting PhIP-DNA adduct formation, CLA does not appear to act by inhibiting CYP 1A1 or 1A2 expression.