Head and neck cancer (HNC) concerns more than 890,000 patients worldwide annually and is associated with the advanced stage at presentation and heavy outcomes. Alcohol drinking, together with tobacco smoking, and human papillomavirus infection are the main recognized risk factors. The tumorigenesis of HNC represents an intricate sequential process that implicates a gradual acquisition of genetic and epigenetics alterations targeting crucial pathways regulating cell growth, motility, and stromal interactions. Tumor microenvironment and growth factors also play a major role in HNC. Alcohol toxicity is caused both directly by ethanol and indirectly by its metabolic products, with the involvement of the oral microbiota and oxidative stress; alcohol might enhance the exposure of epithelial cells to carcinogens, causing epigenetic modifications, DNA damage, and inaccurate DNA repair with the formation of DNA adducts. Long-term markers of alcohol consumption, especially those detected in the hair, may provide crucial information on the real alcohol drinking of HNC patients. Strategies for prevention could include food supplements as polyphenols, and alkylating drugs as therapy that play a key role in HNC management. Indeed, polyphenols throughout their antioxidant and anti-inflammatory actions may counteract or limit the toxic effect of alcohol whereas alkylating agents inhibiting cancer cells’ growth could reduce the carcinogenic damage induced by alcohol. Despite the established association between alcohol and HNC, a concerning pattern of alcohol consumption in survivors of HNC has been shown. It is of primary importance to increase the awareness of cancer risks associated with alcohol consumption, both in oncologic patients and the general population, to provide advice for reducing HNC prevalence and complications.

Ethyl glucuronide (EtG) is a minor, non-oxidative ethanol metabolite detectable in several matrices for specific periods of time. In recent years, quantification of EtG in hair has been established as the most reliable biomarker for long-term alcohol consumption, with the Society of Hair Testing offering cut-off values for assessment of both abstinence and heavy drinking. Instrumental constrains and wide inter- and intra-laboratory variability represent the ultimate barriers to widespread acceptance of hair EtG determination in the forensic context. In this study, a new analytical method for hair EtG based on gas chromatographic (GC) separation, electron impact (EI) ionization, and tandem mass spectrometry (MS/MS) detection was developed and validated. At the same time, several parameters for sample pretreatment and instrumental analysis were optimized using real hair samples obtained from different drinking subjects. A full-factorial design-of-experiment approach included procedures for hair washing, pulverization, and extraction. Rigorous multi-step washing proved not to reduce the EtG content extracted in the subsequent sample incubation. Hair pulverization with a ball mill significantly improved the EtG extraction from the keratin matrix and allowed us to reduce the time needed for the subsequent extraction step, without affecting the extraction recovery. The hair extract was derivatized with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. Upon electron impact ionization of the EtG-TMS derivative, triple quadrupole mass analyzers were operated in the selected reaction monitoring (SRM) mode using the fragment m/z 405 as the precursor ion (m/z 410 for the EtG-D5 internal standard), the transitions m/z 405 → 359 and m/z 410 → 359 for quantitation, and m/z 405 → 331 and m/z 405 → 287 for qualification/confirmation, all at 10 V collision energy. The final method was fully validated and then applied to 25 real hair samples. The calibration curve proved linear between 6 and 60 pg/mg. The limit of detection (LOD) was 4 pg/mg. Intra- and inter-assay precision and accuracy tests showed a variability and bias close to 15% or lower over the entire calibration range. The new method is routinely applied in the Italian State Police’s toxicology laboratory for hair analyses addressed to exclude excessive alcohol drinking and verify the psycho-physical requirements of the personnel.

Alcohol consumption is associated with oxidative stress and an increased risk of carcinoma of the upper aero-digestive tract (UADT). Recently, it has been found that some microorganisms in the human oral cavity may locally metabolize ethanol, forming acetaldehyde, a carcinogenic metabolite of alcohol. In a cohort of patients first visited for UADT cancers, we estimated their alcohol consumption by measuring Ethyl Glucuronide/EtG (a long-lasting metabolite of ethanol) in the hair and carbohydrate-deficient transferrin/CDT (short-term index of alcohol intake) in the serum. Moreover, we analyzed, by culture-based methods, the presence of Neisseria subflava, Streptococcus mitis, Candida albicans, and glabrata (microorganisms generating acetaldehyde) in the oral cavity. According to the EtG values, we correlated drinking alcohol with endogenous oxidative stress and the investigated microorganism’s presence. We found that 55% of heavy drinkers presented microorganisms generating acetaldehyde locally. Moreover, we found that the presence of oral acetaldehyde-producing bacteria correlates with increased oxidative stress compared to patients without such bacteria. As for the study of alcohol dehydrogenase gene polymorphisms (the enzyme that transforms alcohol to acetaldehyde), we found that only the “CGTCGTCCC” haplotype was more frequent in the general population than in carcinoma patients. This pilot study suggests the importance of estimating alcohol consumption (EtG), the presence of bacteria producing acetaldehyde, and oxidative stress as risk factors for the onset of oral carcinomas.