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.