General toxicity and genotoxicity of alternariol: a novel 28-day multi-endpoint assessment in male Sprague-Dawley rats.

Alternariol (AOH) is one of the toxins of Alternaria, and it has been widely detected in a variety of foods. It has been reported to be cytotoxic, dermally toxic, genotoxic, and potentially carcinogenic in vitro. However, in vivo toxicity data are lacking. This study used a novel in vivo 28-day multi-endpoint (Pig-a assay + micronucleus test + comet assay) genotoxicity evaluation system to evaluate the general toxicity and genotoxicity of AOH. A total of 42 male Sprague-Dawley rats were randomly distributed into three AOH-treated groups (5.51, 10.03, and 22.05 µg/kg bw), one AOH high-dose recovery group (AOH-HR, 22.05 µg/kg bw), one positive control group (N-ethyl-N-nitrosourea, 40 mg/kg bw), and two vehicle control groups (corn oil and PBS). Treatments were administered by oral gavage for 28 consecutive days. Histopathological lesions were observed in the liver, kidney, and spleen in all AOH-treated groups. No statistical difference was found in each genotoxicity index within 28 days in the AOH-treated groups compared with those in the corn oil group. On day 42, in the AOH-HR group, the rate of Pig-a mutant phenotype reticulocytes (RETCD59-) significantly increased. On day 56, both RETCD59- and the rate of Pig-a mutant phenotype erythrocytes (RBCCD59-) were significantly reduced. These findings indicated that AOH might cumulatively induce genetic mutations.

Determination of potential thresholds for N-ethyl-N-nitrosourea and ethyl methanesulfonate based on a multi-endpoint genotoxicity assessment platform in rats.

The main goal of the study was to investigate the genotoxic response of N-ethyl-N-nitrosourea (ENU) and ethyl methanesulfonate (EMS) at low doses in a multi-endpoint genotoxicity assessment platform in rats and to derive potential thresholds and related metrics. Male Sprague-Dawley rats were treated by daily oral gavage for 28 consecutive days with ENU (0.25 ~ 8 mg/kg bw) and EMS (5 ~ 160 mg/kg bw), both with six closely spaced dose levels. Pig-a gene mutation assay, micronucleus test, and comet assay were performed in several timepoints. Then, the dose-response relationships were analyzed for possible points of departure (PoD) using the no observed genotoxic effect level and benchmark dose (BMD) protocols with different critical effect sizes (CES, 0.05, 0.1, 0.5, and 1SD). Overall, dose-dependent increases in all investigated endpoints were found for ENU and EMS. PoDs varied across genetic endpoints, timepoints, and statistical methods, and selecting an appropriate lower 95% confidence limit of BMD needs a comprehensive consideration of the mode of action of chemicals, the characteristics of tests, and the model fitting methods. Under the experimental conditions, the PoDs of ENU and EMS were 0.0036 mg/kg bw and 1.7 mg/kg bw, respectively.

Characterization of benzo[a]pyrene and colchicine based on an in vivo repeat-dosing multi-endpoint genotoxicity quantitative assessment platform.

Two prototypical genotoxicants, benzo[a]pyrene (B[a]P) and colchicine (COL), were selected as model compounds to deduce their quantitative genotoxic dose-response relationship at low doses in a multi-endpoint genotoxicity assessment platform. Male Sprague-Dawley rats were treated with B[a]P (2.5-80 mg/kg bw/day) and COL (0.125-2 mg/kg bw/day) daily for 28 days. The parameters included were as follows: comet assay in the peripheral blood and liver, Pig-a gene mutation assay in the peripheral blood, and micronucleus test in the peripheral blood and bone marrow. A significant increase was observed in Pig-a mutant frequency in peripheral blood for B[a]P (started at 40 mg/kg bw/day on Day 14, started at 20 mg/kg bw/day on Day 28), whereas no statistical difference for COL was observed. Micronucleus frequency in reticulocytes of the peripheral blood and bone marrow increased significantly for B[a]P (80 mg/kg bw/day on Day 4, started at 20 mg/kg bw/day on Days 14 and 28 in the blood; started at 20 mg/kg bw/day on Day 28 in the bone marrow) and COL (started at 2 mg/kg bw/day on Day 14, 1 mg/kg bw/day on Day 28 in the blood; started at 1 mg/kg bw/day on Day 28 in the bone marrow). No statistical variation was found in indexes of comet assay at all time points for B[a]P and COL in the peripheral blood and liver. The dose-response relationships of Pig-a and micronucleus test data were analyzed for possible point of departures using three quantitative approaches, i.e., the benchmark dose, breakpoint dose, and no observed genotoxic effect level. The practical thresholds of the genotoxicity of B[a]P and COL estimated in this study were 0.122 and 0.0431 mg/kg bw/day, respectively, and our results also provided distinct genotoxic mode of action of the two chemicals.

General toxicity and genotoxicity of altertoxin I: A novel 28-day multiendpoint assessment in male Sprague-Dawley rats.

The mycotoxin altertoxin I (ATX-I) is one of secondary metabolites produced by Alternaria fungi and is frequently detected as food and feed contaminants. Little is known about the genotoxicity of the ATX-I. In order to evaluate potential genotoxicity and general toxicity of ATX-I, the novel 28-day multiendpoint (Pig-a assay + micronucleus [MN] test + comet assay) genotoxicity platform was applied. Male Sprague-Dawley (SD) rats were randomized to five groups (six rats per group), that is, a positive control group (N-ethyl-N-nitrosourea [ENU], 40 mg/kg.bw/d), two solvent control groups (PBS and corn oil), and two ATX-I-treated groups (low-dose group [1.10 µg/kg.bw/d] and high-dose group [5.51 µg/kg.bw/d]). Treatments were administered by oral gavage to male SD rats for 28 consecutive days. Histopathological damages in the liver, kidney, and spleen were observed, but without significant changes in hematological and serum biochemical parameters. Genotoxic endpoints indicated that ATX-I could cause DNA damage. To summarize, in a relatively low-dose range, ATX-I may not have direct genotoxicity in vivo but could induce liver, kidney, and spleen damage.

Alternariol monomethyl ether toxicity and genotoxicity in male Sprague-Dawley rats: 28-Day in vivo multi-endpoint assessment.

Alternariol monomethyl ether (AME), a typical Alternaria toxin, has often been detected in grains. We have measured the general toxicity and genotoxicity of AME with a 28-day multi-endpoint (Pig-a assay + in vivo micronucleus [MN] test + comet assay) platform. Male Sprague-Dawley rats were administered AME (1.84, 3.67, or 7.35 µg/kg body weight/day), N-Ethyl-N-nitrosourea (40 mg/kg body weight/day), or corn oil by gavage for 28 consecutive days. Another group (AME-high-dose + recovery) was maintained for a further 14 days after the end of the AME administration. Hematology and serum biochemistry results suggested that AME might compromise the immune system. The histopathology results indicated that AME can cause liver (inflammatory cell infiltration, steatosis, and edema), kidney (renal glomerular atrophy), and spleen (white pulp atrophy) damage. The genotoxicity results showed that AME can induce gene mutations, chromosome breakage, and DNA damage, but the effects were diminished after the recovery period. According to point-of-departure analysis (BMDL10), the risk to the population of exposure to AME cannot be ignored and further assessment is needed.

Genotoxicity of three mycotoxin contaminants of rice: 28-day multi-endpoint assessment in rats.

Deoxynivalenol (DON), zearalenone (ZEN), and fumonisin B1 (FB1), as the main mycotoxins contaminating rice, often coexist in food. Thus, we have measured the genotoxicity of the three rice fungal contaminants, singly and in different combinations, with a 28-day multi-endpoint (Pig-a assay + in vivo micronucleus [MN] test + comet assay) genotoxicity platform. Male Sprague-Dawley rats received the agents orally via gavage for 28 consecutive days, before performing the abovementioned tests. Results indicated that low dose of a single mycotoxin did not show significant genotoxicity. However, some of these mycotoxins in combination induced significant genotoxicity in the peripheral blood and tissues, at sacrifice. In the peripheral blood, the binary combination of DON and FB1 significantly induced MN. In the liver, ZEN might aggravate the DNA-damaging effects of DON and FB1. Therefore, the genotoxicity of sub-chronic exposure to mycotoxins in combination cannot be ignored.

Bioinformatics analysis and quantitative weight of evidence assessment to map the potential mode of actions of bisphenol A.

Bisphenol A (BPA) is a classical chemical contaminant in food, and the mode of action (MOA) of BPA remains unclear, constraining the progress of risk assessment. This study aims to assess the potential MOAs of BPA regarding reproductive/developmental toxicity, neurological toxicity, and proliferative effects on the mammary gland and the prostate potentially related to carcinogenesis by using the Comparative Toxicogenomics Database (CTD)-based bioinformatics analysis and the quantitative weight of evidence (QWOE) approach on the basis of the principles of Toxicity Testing in the 21st Century. The CTD-based bioinformatics analysis results showed that estrogen receptor 1, estrogen receptor 2, mitogen-activated protein kinase (MAPK) 1, MAPK3, BCL2 apoptosis regulator, caspase 3, BAX, androgen receptor, and AKT serine/threonine kinase 1 could be the common target genes, and the apoptotic process, cell proliferation, testosterone biosynthetic process, and estrogen biosynthetic process might be the shared phenotypes for different target organs. In addition, the KEGG pathways of the BPA-induced action might involve the estrogen signaling pathway and pathways in cancer. After the QWOE evaluation, two potential estrogen receptor-related MOAs of BPA-induced testis dysfunction and learning-memory deficit were proposed. However, the confidence and the human relevance of the two MOAs were moderate, prompting studies to improve the MOA-based risk assessment of BPA.

[Assessment of the genotoxicity of 2-methylfuran based on a multi-endpoint genotoxicity test system in vivo].

OBJECTIVE: This study was designed to determine the genotoxicity of 2-methylfuran based on a multi-endpoint genotoxicity test system. METHODS: The SPF-grade male SD rats(n = 30) were randomized to six treatment groups, i. e. 4 treatment groups(25, 50, 100 and 150 mg/kg), a control group(vegetable oil) and a positive groups(N-ethyl-N-nitrosourea, 80 mg/kg). All treatments were administrated by gavage for continuous 3 days. Tail vein blood for comet assay was collected at 3 h after the final administration. Pig-a gene mutation assays were performed on days 0(one day before gavage), 14 and 28. Micronucleus tests in peripheral blood using flow cytometry were performed on days 0 and 4. RESULTS: A statistically significant increase in tail intensity was observed at 150 mg/kg for peripheral blood in comet assay. There was no significant difference among the groups in mutant cell frequency of erythrocytes and reticulocytes at 2 timepoints in Pig-a gene mutation assay, and no significant difference among the groups in the frequency of micronucleus in micronucleus test. CONCLUSION: The result of genotoxicity tests suggested that 2-methylfuran was probably not mutagenic in vivo after acute exposure.

An updated weight of evidence approach for deriving a health-based guidance value for 4-nonylphenol.

4-Nonylphenol (NP) is a persistent estrogen-active compound. Human exposure to NP is primarily through water and food. Although risk assessments of NP have been conducted by the European Union and a few other countries, only the Danish Veterinary and Food Administration, in 2000, proposed a tolerable daily intake of 0.005 mg kg-1 body weight (bw) day-1 . New data have been accumulated since then, prompting an update on the risk assessment of NP. A weight of evidence approach is recommended for use in scientific assessments by several agencies, e.g., European Food Safety Authority, etc. Based on the results of a weight of evidence approach, two methods were used to derive the health-based guidance value (HBGV) for NP in this study, namely a no observed adverse effects level/lowest observable adverse effect level method, and a benchmark dose method. Considering the considerable uncertainty of benchmark dose model fitting of the available data, a tolerable daily intake value of 0.025 mg kg-1 bw day-1 was derived as a provisional HBGV for NP based on the lowest observable adverse effect level value of 15 mg kg-1 bw day-1 of the renal toxicity in rats, together with the uncertainty factor of 600. However, the HBGV of NP still needs further investigation.

Dietary cadmium exposure assessment in rural areas of Southwest China.

Dietary exposure of cadmium (Cd) has not been studied in Southwest China. The objective of the study was to determine the pollution characteristics and contamination levels in various agriculture products in Southwest China and to conduct a comparison of dietary exposure assessment of Cd in polluted and non-polluted areas. Results showed that the mean Cd contents in rice were 0.53 and 0.52 mg/kg in the high-polluted and low-polluted areas, respectively, with the average value was 0.03 mg/kg in the control area. The mean dietary Cd exposure from rice and vegetables of the selected non-occupational residents in Southwest China was 113.10 µg/kg bodyweight (bw)/month, 88.80 µg/kg bw/month, and 16.50 µg/kg bw/month in the high-polluted, low-polluted, and control areas, respectively, which correspond to 4.5 times, 3.6 times, and 0.66 times of the provisional tolerable monthly intake (25 µg/kg bw/month) established by the Joint FAO/WHO Expert Committee on Food Additives. The findings indicated that the risk for Cd exposure of residents was high due to home-grown food (most especially rice) being near polluted areas and is of great concern.

[Optimization ofPig-aGene Mutation in Rats and Assessment of Time-dependent and Dose-response Relationship of N-ethyl-N-nitrosourea].

OBJECTIVES: To optimize the method of Pig-a mutation assay, and to explore the time-dependent and dose-response relationship of N-ethyl-N-nitrosourea (ENU). METHODS: Thirty rats were randomly assigned to 5 groups: treated with PBS (control group)or different doses of ENU (10, 20, 40 and 80 mg/kg) for 3 d by oral gavage. Blood samples were collected at 0 d, 15 d, 30 d, 45 d, 60 d, 75 d and 90 d. After enrichment, erythrocytes were incubated with Anti-CD59-APC and SYTO 13 nucleic acid dye solution. Mutant phenotype erythrocytes (RBCCD59-) and mutant phenotype reticulocytes (RETCD59-) were measured by flow cytometry to analyze mutant frequencies, and the RET percentage was determined as well. RESULTS: The RBCCD59- mutation frequency in 4 ENU groups were significantly increased in a dose- and time-dependent manner. The RETCD59- mutation frequency increased to a stable high level with a slight fluctuation, and decreased at 45 d , with the peak values observed at 30 d. The RETCD59- mutation frequency showed a dose-dependent trend in 4 ENU groups. The RET percentage in all 5 groups declined at 30 d, to a stable low level thereafter, but the trends showed no significant differences by time or group. CONCLUSIONS: The optimized in vivo Pig-a mutation assay could detecte the mutagen, such as ENU, induces mutation in RBC in a time- and dose-dependent manner.

[Genotoxicity assessment of 2-ethylhexyl acrylate using an in vivo Pig-a gene mutation assay integrated system].

OBJECTIVE: To verify the mutagenicity of 2-ethylhexyl acrylate( 2-EHA)using in vivo Pig-a gene mutation assay integrated system. METHODS: The SPF-grade male SD rats( n = 30) were randomized to six treatment groups, i. e. 4 treatment groups( 100, 200, 400 and 800 mg/kg), a control group( vegetable oil) and a positive groups( Nethyl-N-nitrosourea, 10 mg/kg). All treatments were administrated by gavage for continuous 28 days. Tail vein blood specimens for Pig-a gene mutation assay were collected on days 0, 15 and 29. The number of mutant erythrocytes and reticulocytes was acquired by flow cytometer. Tail vein blood for comet assay was collected at 6 h after the final administration, followed by the bone marrow micronucleus test after animal sacrifice. RESULTS: Later in the study, signs of mild poisoning were observed in the animals treated with 400 and 800 mg/kg BW 2-EHA. There was no significant difference among the groups in mutant cell frequency of erythrocytes and reticulocytes at all 3 timepoint in Piga gene mutation assay, and no significant difference among the groups in tail length and Olive tail moment in comet assay. There was likewise no significant difference among groups in micronucleus test. CONCLUSION: In present experiment conditions, 2-EHA did not show genotoxicity in Pig-a gene mutation assay, comet assay and micronucleus test, which indicated that 2-EHA probably is not mutagenic in vivo.