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Abstract

There are more questions than answers in biomarkers research at low exposure levels. The present dissertation aimed to estimate the appropriateness of exposure and effect biomarkers, the impact of single nucleotide polymorphisms (SNPs; 35 SNPs, 20 genes) – as susceptibility/adaptability biomarkers – on potentially toxic trace elements (TE) in spot urine and blood samples, and to obtain SNPs allele frequencies. Associations were estimated and tested by multivariable linear regression with the adjustment for selected confounders. Selected Slovenian, Croatian, and Kyrgyzstan populations with low to moderate environmental or diet TE exposure were chosen regarding their vulnerability related to either pregnancy, lactation, childhood, childbearing age or residential region. Some observations were tested on a smaller group of cancer patients pharmacologically exposed to metalloid-containing medicine.
Results confirmed that the normalization of spot urine biomarkers levels for inter-individual variations in hydration status, particularly by creatinine, is inappropriate at low TE exposure due to a high difference in the measurement sensitivity and specificity between creatinine and TEs, resulting in conflicting and inconsistent results. As such, TE determined in blood (plasma) have found to be a better biomarker of low level exposure. Similarly questionable is normalization of urine biomarkers of renal effect (tubular: alpha-1-microglobulin – A1M, N-Acetyl beta-D-Glucosaminidase – NAG; glomerular: Albumin – Alb, Immunoglobulin G – IgG).
Additionally, the associations were clearly influenced by physiological variability. Cd – renal effect biomarkers associations were driven by the normal physiological changes in kidney function rather than by Cd-induced renal effects. Moreover, renal protein A1M was affected also by factors unrelated to kidney function, such as smoking, pointing on its versatile physiological roles and low specificity, which at such low levels oppose its appropriateness as a biomarker of Cd effects.
Superior effect of physiological non-genetic factors versus genetic factors on TE at low exposures was observed during pregnancy. Pregnancy status silenced the most of studied SNPs/TE interactions. Among non-pregnant women, we identified the “protective” role of AS3MT haplotype, related to As methylation efficiency. In accordance with extremely low environmental and diet related iAs exposure we observed 7 times lower frequency of AS3MT haplotype for the Slovenian-Croatian population than that reported for the population of Argentina with historically high iAs exposure. Assessment of AS3MT haplotype and its mRNA levels in As2O3-treated cancer patients pointed out that the former rather than the latter could be used as a predictor of therapeutic efficiency.
Novel assessment of SNPs of AQP – coding small molecules transport channels – showed significant negative influence of AQP4 (rs9951307) and positive influence of AQP9 (rs2414539) on As methylation efficiency and metabolite excretion – the first one during pregnancy only and the second one during non-pregnancy. INMT (rs6970396) and AQP9 both resulted, beside As, also in higher urine levels of Se pointing on their co-excretion, maybe as an As-Se complex. At moderate Pb exposures, we also confirmed influences of two ALAD SNPs (rs1805313 and rs1800435) on lower blood Pb. Frequencies of ALAD SNPs considerably varied among three studied areas of the Kyrgyzstan population and put in question a possible role of geology and environmental pollution on observed levels. Similar differences are expected for Slovenia after genotyping industrially polluted regions including the Mežica valley (Pb and Zn mine).
The present dissertation contributed to the worldwide data on allele frequencies of 35 SNPs in 20 genes for the Slovenian-Croatian population. Significant influence of AQPs, INMT and ALAD SNPs points on their possible role as susceptibility or adaptability biomarkers in TE exposure. However, their role should be individually and/or in combinations tested also in populations/individuals with higher environmental, occupational and/or therapeutic exposure. Due to the observed influence, Se must be included as an association’s modifier.