Immunotoxicity of aflatoxin B1 in rats: effects on lymphocytes and the inflammatory response in a chronic intermittent dosing study.

We investigated the effects of aflatoxin B1 (AFB1) on isolated splenic lymphocytes and the histo-morphologic changes in the spleens and liver of Fisher-344 male rats. Weaned animals were fed chow diets that contained 0, 0.01, 0.04, 0.4, or 1.6 ppm AFB1, using an intermittent dosing regimen (4 weeks on and 4 weeks off AFB1), for 40 weeks. An additional group of animals was fed the 1.6 ppm AFB1 diet continuously. The intermittent dosing regimen was designed to evaluate effects of cumulative dose and exposure for risk assessment comparisons. The percentages of T and B cells were affected as shown by flow cytometric analysis after the dosing cycles. The observed changes appeared to reverse or compensate to some extent after the off cycles. Lymphocytes were stimulated in culture for analysis of the production of IL-2, IL-1, and IL-6. Significantly increased production of IL-1 and IL-6 was seen in the second dosing cycle (12 weeks) and the second "off" cycle (16 weeks) at the higher doses. Inflammatory infiltrates were seen in the liver after eight weeks of continuous and intermittent dosing and were increased in size and number at 12 weeks in both 1.6 ppm dose groups correlating with the peak production of Il-1 and IL-6. We concluded that AFB1 effects on the immune system can be either stimulatory or suppressive dependent on a critical exposure window of dose and time. Immune cells in spleen such as T-lymphocytes and macrophages, both important mediators of inflammatory responses to tissue damage, were affected differently in the continuous and intermittent exposures to AFB1.     

Effects of intermittent exposure to aflatoxin B1 on DNA and RNA adduct formation in rat liver: dose-response and temporal patterns.

We studied the effects of intermittent exposure to aflatoxin B1 (AFB1) on hepatic DNA and RNA adduct formation. Fisher-344 male rats were fed 0.01, 0.04, 0.4, or 1.6 ppm of AFB1 intermittently for 8, 12, 16, and 20 weeks, alternating with 4 weeks of dosing and 4 weeks of rest. Other groups of rats were fed 1.6 ppm of AFB1 continuously for 4, 8, 12, and 16 weeks. Control rats received AFB1-free NIH-31 meal diet. AFB1-DNA and -RNA adducts were measured by HPLC with fluorescence detection. The data are presented as total DNA or RNA adducts. The DNA and RNA adduct levels increased or decreased depending on the cycles of dosing and rest. Rats removed from treatment 1 month after 1 or 2 dosing cycles (8 and 16 weeks of intermittent exposure) showed approximately a twofold decrease in DNA adduct levels and a two- to elevenfold decrease in RNA adduct levels compared with rats euthanized immediately after the last dosing cycle (12 and 20 weeks of intermittent exposure). Our data indicate that DNA and RNA adducts increased linearly, from 0.01 ppm to 1.6 ppm of AFB1 after 12 and 20 weeks of intermittent treatment. A linear dose response was also apparent for DNA but not for RNA adducts after 8 and 16 weeks of treatment. As biomarkers of exposure, AFB1-RNA adducts were three to nine times more sensitive than AFB1-DNA adducts but showed greater variability. These results suggest that binding of AFB1 to hepatic DNA is a linear function of the dose, regardless of the way this is administered. The dose-response relationship for RNA adducts depends on the length of the no-dosing cycles and on the turnover rate of RNA.     

Effects of prenatal aflatoxin B1 exposure on behaviors of rat offspring.

The effects of prenatal aflatoxin B1 (AFB) exposure on eight behavioral parameters in Jcl:Wistar rat offspring were assessed. Pregnant rats were injected subcutaneously with 0.3 mg/kg/day of AFB dissolved in dimethylsulfoxide on days 11-14 (Group A) or 15-18 (Group B) of gestation. Controls received the vehicle similarly on days 11-18 of gestation. Before weaning, the offspring were examined using the cliff avoidance response (5 days of age), the negative geotaxis reflex (7 days), and swimming development (6, 8, and 10 days). After weaning, animals were examined using the rotarod test (5 weeks of age), the open field test (6 weeks), a conditioned avoidance learning test (14 weeks), an underwater T-maze test (15 weeks), and a reproduction test (16 weeks). The preweaning offspring in the AFB-A group showed significantly lower success rates than controls in cliff avoidance responses. In swimming development, the offspring in the AFB-A group had significantly lower scores than controls for swimming direction. In the rotarod test, the AFB-A group remained on the rod for a significantly shorter time than the controls at 15 rpm on both the first and second trial days. The avoidance performance of the rats in AFB-A and AFB -B groups was significantly poorer than that of controls. These results indicate that prenatal exposure to AFB produced a delay of early response development, impaired locomotor coordination, and impaired learning ability in the offspring of rats exposed to AFB during middle pregnancy, and the early gestational exposure appears to produce more effects than latter exposure.     

金蒲抑瘤片在实验诱发大鼠肝癌过程中的作用

目的探讨金蒲抑瘤片对黄曲霉毒素B1(aflatoxin B1,AFB1)致肝癌作用的影响。方法实验动物随机分为高剂量、低剂量和对照组。用AFB1处理各组动物,高、低剂量组大鼠在接受AFB1期间分别喂含量为9.3和2.3g/kg的金蒲抑瘤片混合饲料,对照组喂基础饲料。8周后处死动物,观察各组动物肝组织内γ-谷氨酰转肽酶阳性肝细胞增生(γ-glu-tamyltranspeptidase-positive hyperplastic livercell,γ-GT)灶的数量和大小。结果高、低剂量金蒲抑瘤片均能减少AFB1诱发的γ-GT灶的数量和大小高、低剂量组的数量分别为0.90和3.72个/cm2,均低于对照组6.10个/cm2,抑制率分别为85%和39%,高剂量组与对照组相比差异有统计学意义,t=2.597,P=0.028。高、低剂量组的大小分别为0.24和1.94mm2/个,均低于对照组2.36mm2/个,抑制率分别为90%和17%,但差异无统计学意义,P>0.05。结论金蒲抑瘤片有减少AFB1诱发大鼠肝γ-GT灶的数量和大小的作用,而高剂量金蒲抑瘤片的减少趋势更强。     

Selective impairment of drug-metabolizing enzymes in pig liver during subchronic dietary exposure to aflatoxin B1.

Consequences of subchronic exposure to aflatoxin B1 (AFB1) on liver monooxygenase and transferase enzymes were compared in control pigs and pigs given 385, 867 or 1,807 microg AFB1/kg of feed for 4 weeks. Animals exposed to the highest dose of toxin developed clinical signs of aflatoxicosis, like liver fibrosis, hepatic dysfunction and decreased weight gain. This group had significantly lower levels of liver cytochrome P450, ethoxyresorufin O-deethylase (EROD) activity, testosterone metabolism, P450 1A and P450 3A protein expression. By comparison, mild degenerative hepatic changes, no hepatic dysfunction but a similar pattern of liver P450 enzymes activity without changes in P450 3A expression were observed in pigs exposed to 867 microg AFB1/kg of feed. Benzphetamine and aminopyrine N-demethylase activities were increased in pigs exposed to 867 or 1,807microg AFB1/kg of feed. Pigs exposed to 385 microg AFB1/kg of feed had low levels of EROD activity and all other biotransformation and clinical parameters remained at control levels. Aniline hydroxylase activity, P450 2C protein expression, UDP-glucuronosyl and glutathione S-transferase activities were unaffected at all doses of AFB1. In conclusion, P450 1A and P450 3A appear to be specific targets of AFB1 even if pig did not display clinical sign of liver toxicosis.     

中药中外源性有害残留物的控制

分析了中药中外源性有害残留的种类及危害、对其研究进展进行了全面回顾,综述了国内外的监控情况。     

Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells following in vivo treatment with aflatoxin B(1).

Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by some strains of Aspergillus and is a recognized pulmonary and hepatic carcinogen. The most widely accepted mechanism of AFB(1) carcinogenicity involves bioactivation to AFB(1)-8,9-exo-epoxide and binding to DNA to form AFB(1)-N(7)-guanine. Another potential cause of DNA damage is AFB(1)-mediated stimulation of reactive oxygen species formation, leading to oxidation of DNA bases. The objective of this study was to determine the ability of AFB(1) to cause oxidative DNA damage in lung cell types of the A/J mouse. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells was assessed by high-performance liquid chromatography with electrochemical detection. An approximately 3-fold increase in 8-OHdG formation occurred in both alveolar macrophage and Clara cell preparations isolated from A/J mice 2 h following treatment with a single tumorigenic dose of 50 mg/kg AFB(1) ip (n = 3, p < 0.05). Prior treatment with 300 kU/kg polyethylene glycol-conjugated catalase prevented the AFB(1)-induced increase in 8-OHdG levels in all mouse lung cell preparations (n = 3, p < 0.05). These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB(1) carcinogenicity.     

肝癌高发区福建同安居民AFTB_1摄入量及膳食营养状况分析

抽取福建同安县新圩和内厝乡６４户居民，对黄曲霉素Ｂ_１（ＡＦＴＢ_１）摄入量及膳食营养状况进行调查采样分析。结果显示，居民常用食品ＡＦＴＢ_１污染较为严重，折合标准成年男子摄入量为２１３６．３ｎｇ／ｄ。某些营养素摄入不足或严重不足，优质蛋白质摄入量为１１．３７ｇ（为标准的１６．２４％）；膳食脂肪摄入３４．６１ｇ；不饱和脂肪酸所占比例较高，尤其是多不饱和脂肪酸；钙、锌、硒摄入量分别为３７４．４５ｍｇ，１１．２１ｍｇ，３６．１７μｇ（为标准的４６．８１％，７４．７３％，７２．３４％）；视黄醇当量及核黄素摄入量分别为１８５．０３μｇ，０．６７ｍｇ；（为标准的２３．１３％和５５．８３％）。同安居民较高的ＡＦＴＢ_１摄入量与原发性肝癌的高发病率有关。     

Genetics of Dothistromin Biosynthesis in the Peanut Pathogen Passalora arachidicola

The peanut leaf spot pathogen Passalora arachidicola (Mycosphaerella arachidis) is known to produce dothistromin, a mycotoxin related to aflatoxin. This is a feature shared with the pine needle pathogen Dothistroma septosporum (Mycosphaerella pini). Dothistromin biosynthesis in D. septosporum commences at an unusually early stage of growth in culture compared to most other fungal secondary metabolites, and the biosynthetic genes are arranged in fragmented groups, in contrast to aflatoxin gene clusters. Dothistromin biosynthetic genes were identified and studied in P. arachidicola to determine if the attributes described in D. septosporum are shared by another dothistromin-producing species within the Class Dothideomycetes. It was shown that dothistromin biosynthesis is very similar in the two species with regard to gene sequence and gene synteny. Functional complementation of D. septosporum mutants with P. arachidicola dothistromin genes was also possible. These similarities support a vertical mode of dothistromin gene transmission. P. arachidicola also produced dothistromin at an early growth stage in culture, suggesting that this type of regulation pattern may be relevant to the biological role of dothistromin.     

Metabolism of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B₁-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB₁ to the exo-AFBO and to detoxification products aflatoxin M₁ (AFM₁) and aflatoxin Q₁ (AFQ₁), respectively, complicates the kinetic analysis of AFB₁ in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB₁ in TLMs. In control TLMs, AFB₁ was converted to exo-AFBO in addition to AFM₁ and AFQ₁ confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~ 0.1 μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 μM) AFB₁ concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM₁, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ₁ following the kinetic Hill equation. At 0.1 μM AFB₁, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM₁) compared to 3A37 (0.15: 1, exo-AFBO: AFQ₁), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB₁ bioactivation and metabolism at environmentally-relevant AFB₁ concentrations in turkey liver.