L.A. Corcuera

Inter-laboratory variation in DNA damage using a standard comet assay protocol

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratorys own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.

Ochratoxin A reduces aflatoxin B1 induced DNA damage detected by the comet assay in Hep G2 cells

Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA+AFB1 for 24 h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo III and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3 h with external metabolic activation (S9 mix) and after 24 h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.

Validation of a UHPLC-FLD analytical method for the simultaneous quantification of aflatoxin B1 and ochratoxin a in rat plasma, liver and kidney.

A rapid and simple method for the simultaneous quantification of AFB1 and OTA in rat plasma, liver and kidney by UHPLC-FLD has been successfully validated according to the following criteria: selectivity, stability, linearity, precision, accuracy, recovery, robustness and limits of quantification and detection. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (100 μL) were extracted with acetonitrile:formic acid mixture (99:1) (300 μL). Chromatographic separation was performed with a mixture of water and acetonitrile:methanol (50:50), both acidified with 0.5% of formic acid using a gradient profile. The method avoids the use of immunoaffinity columns and allows reduction of sample and solvent volumes as well as toxic wastes. The detection is based on a photochemical reaction which enhances the AFB1 response without affecting the OTA signal before reaching the fluorescent detector. The mycotoxin recovery for each matrix was very efficient, between 93% and 96% for AFB1 and between 94% and 96% for OTA. For both mycotoxins the LOQs were 2μg/L in plasma and 8μg/kg in liver and kidney. The method has successfully been applied to rat samples after a single oral administration of a mixture of AFB1 and OTA and it could be a useful tool in toxicokinetic and toxicological studies.

A different kinetic profile of ochratoxin A in mature male rats.

Ochratoxin A (OTA) is a mycotoxin that causes renal tumors in rodents, particularly in male rats. The present work explored the impact of gender and age on OTA toxicokinetics in F344 rats after a single oral dose (0.5mg/kg b.w.). OTA plasma concentrations were analysed with a validated HPLC-FLD method and a population approach (NONMEM VI) was used to perform the kinetic analysis and the one year exposure simulation (0.21 mg/kg daily). Maximum observed OTA concentration (CMAX(obs)) was at 2h in all groups except in mature females (6h). Mature females reached higher CMAX(obs) than males of the same age. Apparent volume of distribution, but not apparent total plasma clearance, increased significantly with body weight (P<0.01) resulting in the following values for the terminal plasma half life (h) in males: 219 (young), 264 (matures) and females: 191 (young), 205 (matures). In addition mature males showed a significant lower relative bioavailability. The simulation showed similar plasma concentrations in males and females after two-months. Thus, toxicokinetic does not seem to explain sex-differences in toxicity in long-term studies. However, the age and weight should be taken into account in short-term toxicological studies if sex-differences are studied.

Genotoxicity of Aflatoxin B1 and Ochratoxin A after simultaneous application of the in vivo micronucleus and comet assay

Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are genotoxic mycotoxins that can contaminate a variety of foodstuffs, the liver and the kidney being their target organs, respectively. The micronucleus (MN) assay (bone marrow) and the comet assay (liver and kidney) were performed simultaneously in F344 rats, treated with AFB1 (0.25 mg/kg b.w.), OTA (0.5 mg/kg b.w.) or both mycotoxins. After AFB1 treatment, histopathology and biochemistry analysis showed liver necrosis, focal inflammation and an increase in Alanine Aminotransferase and Aspartate Aminotransferase. OTA alone did not cause any alteration. The acute hepatotoxic effects caused by AFB1 were less pronounced in animals treated with both mycotoxins. With regard to the MN assay, after 24 h, positive results were obtained for AFB1 and negative results were obtained for OTA, although both toxins caused bone marrow toxicity. In the combined treatment, OTA reduced the toxicity and the number of MN produced by AFB1. In the comet assay, after 3 h, positive results were obtained for AFB1 in the liver and for OTA in the kidney. The combined treatment reduced DNA damage in the liver and had no influence in the kidney. Altogether, these results may be indicative of an antagonistic relationship regarding the genotoxicity of both mycotoxins.

Simple high-performance liquid chromatography-fluorescence detection method for plasma, kidney and liver of rat as a tool for toxicology studies.

A fast and simple HPLC-FLD (high-performance liquid chromatography-fluorescence detection) analytical method has been developed and validated for the determination of ochratoxin A in rat plasma, kidney and liver. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (250 microL) were extracted with ethanol (400 microL) and trichloroacetic acid 20% (w/v) (50 microL). Supernatants were directly injected into the HPLC system, analyzed on a 5-microm (25 cm x 0.4 cm) Tracer Extrasil ODS2 column using FLD (excitation wavelength=225 nm, emission wavelength=461 nm). The mobile phase was 29:29:42 (v/v) methanol-acetonitrile-sodium acetate. The small volume of sample needed which allows the obtaining of ochratoxin A levels in individual tissue samples from small animals and the wide range of concentrations that could be analyzed make this method easy to apply in toxicology and toxicokinetic studies of this mycotoxin, even in low dose carcinogenic studies. This method was linear and selective for all the matrices. Precision and accuracy were always <10% and recovery was very efficient in each case. Limits of detection and quantification were also calculated in plasma (1 and 8.4 microg/L), kidney (14.3 and 55.8 microg/kg) and liver (4.1 and 52.8 microg/kg). Stability of the tissue homogenates was assured for at least 10 months at -80 degrees C. The method has been successfully applied to the analysis of rat samples after 7 days of ochratoxin A (0.5mg/kg b.w. dissolved in an aqueous NaHCO(3) solution) administration by oral gavage.

Kidney and liver distribution of ochratoxin A in male and female F344 rats.

The impact of age and gender on Ochratoxin A (OTA) distribution in kidney and liver were studied. OTA was quantified in kidney and liver of young and mature rats of both sexes. Data was fit simultaneously using the population approach with NONMEM program. Fed and fasted mature males showed a 30% decrease and an 11% increase in relative bioavailability, respectively, in comparison with the rest of the groups. The OTA concentrations reached in kidney and liver were very similar between both organs. The models that best fit to data were the ones that considered that distribution of OTA to kidney and liver occurs from the central compartment and that elimination occurs mainly from the liver compartment. The kinetic analysis revealed that both, the apparent volume of distribution of the central compartment (V/F) and the apparent volume of distribution of the liver and kidney compartments (V(L,K)/F) increased significantly with body weight. Thus, the sex differences observed in organs distribution are a reflection of the differences in relative bioavailability observed in adult males, as a consequence of the fed and fasted conditions and to the significant higher body weight of mature males which directly affected the V/F and V(L,K)/F.

Effects of fasting and gender on ochratoxin A toxicokinetics in F344 rats

Ochratoxin A (OTA) is a mycotoxin that causes renal tumors in rats, particularly in males. In previous kinetic studies performed in fed conditions (Vettorazzi et al., 2008), mature F344 male rats presented a significantly lower OTA bioavailability than females and young animals. The objective of the present study was to evaluate two factors which could explain this different kinetic profile: the presence of food and the male-specific protein alpha-2u-globulin. Therefore, a 24h kinetic study has been performed in rats under fasting conditions. Food ingestion has been controlled in both sexes during two months. The presence of alpha-2u-globulin in the urine has been analyzed with SDS-gradient mini-gel electrophoresis. Fasting tends to increase the maximum OTA plasma concentrations and the rate of absorption. The relative bioavailability is significantly increased under fasting conditions only in males. Mature males consumed a higher amount of food but, as the OTA dose administered, it was proportional to body weight. The reason why the OTA bioavailability is more affected in presence of food only in males is unclear. Several possibilities, such as differences in gastric emptying, OTA-food interactions and the involvement of alpha-2u-globulin are discussed.

An approach to the toxicity and toxicokinetics of aflatoxin B1 and ochratoxin A after simultaneous oral administration to fasted F344 rats.

Humans are exposed to the hepatotoxic aflatoxin B1 (AFB1) and nephrotoxic ochratoxin A (OTA) through diet. However, kinetic and toxicological data after their co-administration are scarce. In this study, a single oral dose of AFB1 (0.25 mg/kg bw)+OTA (0.5 mg/kgbw) was administered to fasted F344 rats. Blood, liver and kidney were harvested at different timepoints for mycotoxins quantification, relative weight calculation, clinical biochemistry and histopathology analysis. Toxicity parameters pointed to acute toxicity in liver due to AFB1. No remarkable toxicity was observed in kidneys or immunological organs. Maximum observed concentrations in plasma (Cmax) were at 10 min and 2 h for AFB1 and OTA, respectively. AFB1 plasma concentration could indicate a rapid absorption/ metabolism of the mycotoxin; and AFB1 liver and kidney concentrations were lower than LOQ and LOD, respectively. For OTA, Cmax was 4326.2 μg/L in plasma. In kidney and liver Cmax was reached at 8 h and concentrations were very similar between both organs at all timepoints. Due to the low levels of AFB1, the effect of OTA on AFB1 kinetics could not be assessed. However, AFB1 seems not to affect OTA kinetics, as its profile seems very similar to kinetic studies performed only with OTA in similar conditions.