Synthesis and in vitro characterization of the genotoxic, mutagenic and cell-transforming potential of nitrosylated heme.

Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis.

Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR.

BACKGROUND: Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed "cassette PCR", in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press "go". Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae, stx1 and stx2). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2. For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145). RESULTS: Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli (eae plus stx1 and/or stx2). Of all samples identified as carrying pathogenic E. coli, 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison. CONCLUSIONS: Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli. Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.

The Potential of Various Living Tissues for Monitoring Clenbuterol Abuse in Food-Producing Chinese Simmental Beef Cattle.

We aimed to evaluate whether living tissues such as urine, plasma and hair were suitable for monitoring clenbuterol (CL) abuse after its subchronic administration of a growth-promoting dose to the Chinese Simmental beef cattle. Eight male, white and red pied Chinese Simmental beef cattle were involved in the experiment, and the CL dose was 16 µg/kg BW/day. Liquid chromatography tandem mass spectrometry (LC-MS-MS) was used to determine CL residues in different tissues, and the addition of D9-clenbuterol internal standard was applied to increase determination accuracy. The recovery of plasma, urine, hair and in vivo tissues was 88.5-114.2, 83.9-114.3, 88.6-116.9 and 85.3-121.7%, respectively. The results showed that CL residue concentrations in the plasma, on Days 14 after withdrawal and later, were lower than the limit of detection (LOD) (0.06 ng/mL) and CL residue in urine was lower than LOD (0.16 ng/mL) 42 days after treatment. CL significantly accumulated in the white and red hair and maintained more than 7.19 ± 2.19 pg/mg within the early withdrawal period of 70 days. A large number of CL were determined in all tested biological tissues, in which residues were higher than the maximum residue limits (MRLs) after dietary administration of CL for 21 days and pre-slaughter withdrawal period of ∼6 h. A particular concern is the slow depletion of residues of CL in some tissues like gluteus and liver still exceeding theirs MRLs, respectively, on Days 14 or 28 days after withdrawal. Our study indicated that plasma and urine could be available for monitoring CL abuse only within a short period of time. However, hair (including light-pigmented) as a target matrix can be selected to perform the long-period monitor of CL.