PIG-A mutation test in human biomonitoring
Other factors that may influence mutation levels in participants include smoking, diet and medication usage. A summary of studies assessing different exposures on erythrocyte PIG-A mutant levels is shown in Table I. Although a significant increase in PIG-A mutant cells was observed in smokers of one study (Haboubi et al., 2019), and an association between the number of PIG-A mutant cells and cigarette-pack-years was seen in another study (Cao et al., 2016), other research groups failed to identify an association between cigarette smoking and mutant cell levels. Whilst the genotoxic nature of cigarette smoking has been extensively studied (Alsaad et al., 2019; Mohammed, Hussen, Rashad, & Hasheesh, 2020), the lack of association withPIG-A mutant frequency may be due to the limited number of smokers recruited in some studies, the lack of information on cigarette-pack-years or the influence of confounding factors including gender, age, diet, inflammation and DNA repair, on mutant cell number.
The effect of diet and lifestyle has been studied in a single, large cohort with results indicating that high dietary intake of vegetables and exercising for more than one hour per week, may reduce mutant cell levels (Lawrence et al., 2020). In addition, aspirin intake may be protective against an increase in PIG-A mutation levels. Whilst the anti-cancer effects of aspirin have been well documented (Jiang, Swacha, Aung, & Gekara, 2023), the specific interaction of dietary intake and erythrocyte mutant levels remains unclear. Cancer treatment i.e., chemo or radiotherapy, may also change mutation levels. One study measured granulocyte PIG-A mutant levels in breast cancer patients undergoing radiotherapy but found no change in mutant cell number. However, the authors state that as the irradiation administered concerned only a part of the sternum and ribs, the probability of a granulocyte precursor gaining a mutation could have been too low to significantly impact blood cell PIG-A mutation levels (Bonetto et al., 2021). Another study measuring erythrocyte mutant cell levels in a cohort of patients undergoing chemotherapy +/- radiotherapy could not evaluate the effect of treatment as no pre-treatment blood samples were obtained. Furthermore, these patients had various types of malignancy and were undergoing different treatment regimens (Horibata, Ukai, Ishikawa, Sugano, & Honma, 2016). Another study identified only 1/10 patients undergoing chemotherapy had a notable (3-fold) increase in erythrocyte PIG-A mutant cell levels (Dobrovolsky et al., 2011). Reticulocyte PIG-A mutant cell levels have been shown to increase in testicular cancer patients (n=3) after cisplatin-based treatment, but it took longer to observe PIG-A mutant reticulocytes compared to micronucleated reticulocytes (Torous et al., 2023). Although we would expect systemic genotoxic agents such as chemotherapy to induce mutant levels, the time between treatment and mutant manifestation is critical. One of the downfalls in the literature to date in measuring PIG-Amutation in patients undergoing treatment, is the lack of pre-treatment samples to allow for meaningful comparisons. In addition, lack of associations between chemotherapy and mutant cell levels may be due to the long-term nature of PIG-A mutant manifestation, where the genotoxic effect is not detectable within the study timeframe (Dobrovolsky et al., 2011). By maximising study durations and obtaining samples at the optimum time point (considering cell life span and/or maturation time from haematopoietic stem cell), this assay could potentially inform us as to which patients are more sensitive to treatment and may therefore respond better to therapy. A test battery would also be most beneficial in measuring chemotherapy sensitivity, as effects may be observed sooner with the micronucleus assay than thePIG-A test, which in turn may be more informative on the accumulation of genotoxic exposure and long-term effects (Torous et al., 2020).
Work carried out by Cao and colleagues has assessed the DNA damage potential of multiple exposures including azathioprine treatment in a cohort of inflammatory bowel disease (IBD) patients (Cao, Wang, Liu, et al., 2020) and occupational exposure to lead (Cao, Wang, Xi, et al., 2020), polycyclic aromatic hydrocarbons (PAHs) (Cao et al., 2021; Xi et al., 2023) and benzene (Cao et al., 2023). Whilst IBD patients had higher mutant cell levels than healthy controls, azathioprine treatment did not affect erythrocyte PIG-A mutant frequency (Cao, Wang, Liu, et al., 2020). Interestingly, PIG-A mutant frequency was higher in workers occupationally exposed to lead (Cao, Wang, Xi, et al., 2020), PAHs (Cao et al., 2021) and benzene (Cao et al., 2023) (compared to controls). The important work carried out in workers exposed to benzene demonstrated that even below the occupational exposure limit (OEL) of 1ppm, workers had elevated levels of erythrocyte PIG-Amutant cells and lymphocyte micronuclei compared to controls, with the authors proposing a new OEL of 0.07ppm (Cao et al., 2023). This study highlights the limitations of current DNA damage tests particularly in the occupational exposure setting. In addition, measuring urinary or airborne levels of genotoxins in this manner should be widely implemented in studies where the exposure is known, with these biomarkers of exposure complementing the PIG-A biomarker of effect. In order to avoid exposing workers to such health hazards, thePIG-A test should be included in a battery of genotoxicity assays as well as quantifying genotoxin levels to re-define OELs and ensure safe work environments. Whilst these tests will be useful as biomarkers of exposure, the potential long-term carcinogenic effects can be elucidated through longitudinal epidemiological studies.
The importance of studying large cohorts is paramount when developing a biomonitoring tool. Disparate or non-reproducible results in some publications could be due to the small number of individuals studied. Whereas studies examining PIG-A MF in hundreds of patients may be more informative, population scale studies including multi-ethnicities, wide age ranges and different exposures (diet, cigarette smoking, medication and occupational genotoxins) are required. These studies will require an international, collaborative effort, but are crucial to the understanding of what drives mutant cell levels, and a necessity for biomarker implementation.
Table I: The effect of different exposures on phosphatidylinositol glycan class A (PIG-A ) mutant frequency.