In vitro genotoxicity assessment of functional ingredients and fried food extracts

Resumen

Food is the main source of human exposure to chemicals, which can be deliberately added or appear as contaminants. In order to protect the consumers, the safety of these chemicals is strictly evaluated, in which genetic toxicology plays an essential role. This doctoral thesis has focused on the in vitro genotoxicity evaluation of functional ingredients and fried food. The bioactive nature of functional ingredients and their alleged beneficial impact in human health arouses a growing commercial interest for both manufacturers and consumers. Although these substances are not specifically regulated, they are marketed under different food regulations in order to guarantee their safety. The in vitro genotoxicity of six compounds widely used as functional ingredients has been assessed: betaine, choline, taurine, docosahexaenoic acid (DHA), rutin and α-tocopherol. A miniaturized version of the Ames test in Salmonella typhimurium following the principles of Organization for Economic Cooperation and Development countries (OECD) 471 guideline, to detect gene mutations, and the in vitro micronucleus (MN) test in TK6 cells (OECD 487 guideline), to detect chromosome aberrations, were performed. The strategy that combines the Ames test with the MN assay is recommended by the European Food and Safety Authority (EFSA) for the in vitro genotoxicity assessment of food and feed. In addition, this approach was complemented with the in vitro enzyme-modified comet assay with and without external metabolic activation using the enzymes human 8-oxoguanine DNA glycosylase 1 (hOGG1), endonuclease III (EndoIII) and human alkyladenine DNA glycosylase (hAAG) in order to assess potential premutagenic lesions in TK6 cells. Betaine, choline, taurine and α-tocopherol did not produce any signs of genotoxicity in any of the conditions tested. Rutin showed an equivocal response in the in vitro MN test and it was a potent S. typhimurium His+ revertant inductor. DHA showed equivocal results in the in vitro MN test. These outcomes complement the available evidence about the genotoxicity of these compounds. The relevance of food processing in the formation of potentially carcinogenic compounds has gained interest in the last twenty years. In fact, processed and red meat have recently been classified as carcinogenic and probably carcinogenic to humans, respectively. In the current doctoral thesis, a systematic review of the genotoxicity of fried meat was conducted. Most articles assessed the mutagenicity of the extracts using the Ames test; they were consistently positive in strains TA98/TA1538 with metabolic activation and few evaluated meat samples from restaurants. To overcome this limitation, the in vitro mutagenicity of ten fried meat-based food, obtained from different catering companies from Navarra (Spain), was evaluated. A miniaturized 6-well version of the Ames test in S. typhimurium TA98, with or without metabolic activation, and the in vitro MN test (OECD 487 guideline) in the thymidine kinase heterozygote cell line (TK6) were performed. None of the ten extracts of fried meat-based food induced gene mutations but five induced chromosomal aberrations after 24 h treatment. Two decades ago, acrylamide, a known in vivo carcinogen, was identified as a process contaminant formed during cooking potatoes. The few studies that have assessed the genotoxicity of French fries showed weak or no in vitro mutagenic activity. However, only the induction of gene mutations was tested. In this regard, the in vitro mutagenicity of French fries obtained from ten different catering companies from Navarra (Spain) was evaluated by the in vitro micronucleus test (OECD 487). Eight out of ten samples induced chromosomal aberrations. Moreover, French fries deep-fried at increasing times (0, 3, 5, 10, 20 and 30 min) in the laboratory induced MN from 3 min on.