To our knowledge, our study is the first meta-analysis and systematic review in the relevant field to incorporate all relevant cohort studies exploring the association of PFOA with liver enzyme indicators. The results of our cohort-based study support the hypothesis that PFOA increases the levels of four common liver enzymes, and this causal relationship was observed to the extent of the population-based study field. However, based on subgroup and descriptive analyses, we still detected that our included studies did not provide consistent strength of evidence support, and the heterogeneity of results across liver enzyme indicators and differences in the degree of adjustment for confounders were limiting factors in the interpretation of causality from our results (Table 4).
The hepatotoxic effects of PFOA in rodents leading to hepatomegaly and hepatocyte histological alterations have been well documented [43, 44], and although different mechanisms also play a role in humans, the PPAR-α agonist-mediated pathway remains one of the main mechanisms leading to altered expression of genes involved in peroxisome proliferation, cell cycle control and apoptosis, and this PPAR-α response has similarly predicted in human cellular responses [45–47]. Exposure to PFOA resulted in site-specific DNA methylation of the mTOR pathway inhibitor Pten gene, decreased gene expression and increased expression of Mtor and Kit, promoting apoptosis and liver injury [26]. The experiments with HepG2 cells cultured in vitro revealed that PFOA activated lipid metabolism genes, altered cellular metabolic rate, increased lipid deposition in hepatocytes, and exhibited great cytotoxicity [28]. PFOA disrupted the activity of metabolic detoxification enzyme CYP450 by blocking the interaction of nuclear translocation complexes with DNA sequences, affecting the conversion and excretion of toxic substances, which may lead to diminished hepatic detoxification [48]. One important mechanism of PFAS interference with fatty acid metabolism and lipid transport is the strong affinity for hepatic fatty acid binding proteins, showing intrinsic hepatotoxicity and bioaccumulation [49, 50].
Several studies have shown that PFOA is a specific risk factor with hepatotoxicity that affects the synthesis and metabolism of liver enzymes, and these studies compared associations between different indicators of liver functional impairment (e.g., liver enzymes, bilirubin, and abnormal lipid metabolism) at different levels of PFOA exposure, but most of this evidence comes from cross-sectional studies as well as cohort studies with small samples, there is a gap in systematic reviews and meta-analyses of longitudinal studies that quantify these associations. Similar to our results, [9, 15, 29] reported that the relationship between PFOA exposure and ALT elevation (a proxy for hepatocyte injury) in longitudinal studies was consistent across analyses, specifically for for PFOA continuous consideration (per one ln-unit increase) and quintile (from the first to the fifth quintile) for cumulative and year-specific serum PFOA, as well as for ALT continuous consideration (ALT level) or as dichotomous results (odds of above-normal ALT), significant relationships of increasing ALT were observed. In contrast, however, studies by [12, 14, 38] suggested no clinical hepatotoxicity associated with PFOA levels, in contrast to previously reported observations. For AST, the existing conclusions of individual cohort studies were not harmonized, with three studies reporting that continuous exposure (per one unit or ln-unit) to PFOA brought a significant increase from 0.25% [9] to 2.9% [13], while three other studies failed to reach a significance result [14, 29, 40]. The majority (n = 4) of the six studies that currently examined PFOA and GGT reported the significant relationship with continuous exposure to PFOA [9, 10, 13, 29], while two other studies that examined the of the effects of community PFAS exposure similarly gave positive effect sizes (0.003 and 0.000) although without statistical significance [15, 40]. Among the four longitudinal studies that focused on the association between PFOA and ALP, only one study reported the significant association between continuous per one unit exposure to PFOA and an increase in ALP at the 0.73% level [13], and two studies respectively from the United States and China failed to generate a statistically significant association [9, 40], and [14] even reported a negative association of continuous per one ln-unit exposure to PFOA with a 17.73% (-1.1750, -0.0007) decrease in ALP levels.
It is noteworthy that substantial and statistically significant heterogeneity was detected in the results of the available cohort analysis for the remaining three of our four liver enzyme indicators, except for ALP (n = 4, 0.007, I-squared = 0.0%) (Table 3), and that there is substantial and inevitable heterogeneity in studies of liver function biomarkers related to environmental exposures that must be considered, as suggested by [30, 51]. Between-group heterogeneity may arise from the dose of PFOA exposure, statistical methods, different sample sizes, inconsistencies in geographic characteristics and the degree of adjustment for confounding factors (e.g., gender, age, alcohol intake, BMI, smoking history, etc.). This limits to some extent the interpretation of our results in terms of implications.
For the effects of combined exposure to different doses of PFOA, while higher PFAS concentrations are always associated with higher ALT in most cross-sectional studies [10, 11, 14, 15, 29, 32], prospective studies have revealed the most null association [32, 52], it also indeed leads to differences in results, as described above [9, 53]. There is still no consensus regarding the shape of the dose-response relationship curve between PFOA and liver enzyme indicators [10, 12, 15, 32], Kennedy et al. characterized sub-chronic and chronic toxicity studies in rodents, revealing the liver as the most sensitive target organ for PFOA action and the dose-dependent increase in serum ALP, ALT, and AST levels associated with PFOA administration [54], which has been verified in many animal experiments [43, 44, 55], however unlike rats and mice, no similar situation was observed in cynomolgus monkeys after oral dosing PFOA (6 months) [56]. While in population experiments, chronic exposure to community PFOA exposure among 371 residents from households who had resided in the Little Hocking Water Association district ([40], PFOA:354(184–571)/(ng/ml)), which is substantially higher than the average PFOA currently observed in general population samples in the United States, but they failed unexpectedly to find any significant positive relationship between serum (PFOA) and markers of liver function (including three classes of liver enzymes) and other markers with potential health effects; In another longitudinal evaluation of clinical parameters with PFOA levels (median) greater than 10 ng/ml, similarly no adverse correlation was found between changes in clinical chemistry of PFOA (50.9 ng/ml), non-HDL cholesterol, HDL and liver enzyme indicators [14]; Another cohort-study from a high-tech fluorescent chemical industrial zone in Jiangsu, China, showed a different conclusion, with Wang et al. [38] measuring median serum PFOA and PFOS levels of 284.34 ng/mL and 34.16 ng/mL in residents and 1635.96 ng/mL and 33.46 ng/mL in occupational participants, after adjusting for confounding, the significant increasing correlation of PFOA with AST was observed in workers (0.2000, 0.1000 to 0.3000), but not in ALT (-0.1700, -0.1900 to 0.0000). Possible explanations for the discrepancy in their conclusions are strongly associated with their insufficient adjustment for confounding factors (especially some major confounders such as alcohol intake, smoking history, age, etc.) and their low RoB scores in all six domains including selection bias, making the quality of their evidence seriously dubious. The other five included cohort studies all measured PFOA levels below 10ng/ml and showed the positive and significant correlation between PFOA exposure and ALT, except for the study by [12] (-0.3000, -0.7333 to 0.1667 ); in ALP, [9] and [13] reported contradictory findings (-0.0111, -0.0336 to 0.0119 versus 0.0073, 0.0028 to 0.0118), respectively; in AST, except for [29] (0.18, -0.0400 to 0.4000), the other two studies reported significant increases; in GGT, the significant relationship was reported in four studies except for [15] (0.0030, -0.0020 to 0.0080). In the absence of explicit prior statements about the dose-response relationship between PFOA and any sort of liver enzyme indicator, this variability strongly suggests a possible nonlinear relationship between them with stronger effects at low dose exposures, but further detailed studies are required.
Effect estimates in our meta-analysis are reported in describing comparisons per one unit or ln-unit change, mainly considering that the available data are mainly clustered under this group. More importantly, Kerger et al. [57] demonstrated that classification by chemical dose level, such as utilizing the low exposure group as a valid control group for statistical comparisons of common disease states, raises significant difficulties in toxicological studies, possibly due to inadvertent selection bias that may have affected the lowest exposure quartile (control group), making the dose-response relationship between PFOA/PFOS and risk of outcome tenuous; also, we have attempted dose-response meta-analysis to investigate a possible dose-response relationship between PFOA and liver enzyme indicators, but the existing number of cohort studies was insufficient to extract the necessary data to proceed. Therefore, in aggregate, our approach is the most reasonable allowing consistent comparison of study-specific estimates and interpretation of findings at this stage.
In addition to the dosage differences mentioned above, which may account for much of the heterogeneity, regional socioeconomic status (SES) or potential sociodemographic predictors have emerged as potentially important confounding factors in environmental health studies [58], 7 of the 9 studies we ultimately analyzed for data synthesis were from the United States, with the exception of 2 from China, by simply classifying we can observe that the studies from China showed statistically significant associations for all three indicators (ALT, AST, GGT) except ALP, which can be moderately explained by the lower foundational health conditions, while the presence of publication bias for positive results must also be considered; the studies we included ranged from the largest sample size of 32254 [15] to the smallest of 74 [13], the effect of sample size on the results was not demonstrated explicitly in our subgroup and main analyses; other factors such as age and gender, given the small amount of evidence available for analysis, subgroup analyses cannot ensure the number of per group greater than 3, which is insufficient to account for specific associations. Given the inherent limitations of epidemiological data, these estimates need to be interpreted with caution, although our study considers only the results of longitudinal cohort studies, which can show associations with respect to time and exclude the possibility of reverse causality to some extent [9, 14, 38].
Gender differences in the association between PFAS and liver enzymes have barely been reported in the literature [15], and only one study of the nine cohorts we included attempted to explore differences between men and women by performing subgroup analyses by gender, Mora et al. [12] reported a prospective Boston area prenatal cohort that revealed in girls during childhood, higher levels of PFOS, PFOA concentrations were associated with deleterious changes (higher TC and/or LDL-C, higher HDL-C, and slightly lower ALT), unlike the differences in PFAS-lipid associations between males and females in different age groups [59–61] which have been widely reported. In the majority of cross-sectional as well as cohort studies, no clear relationship between PFOA exposure level and gender was found [15], but [38] reported differences in the gender distribution of two PFAS (PFTA and PFHxS), specifically higher dosages of exposure were observed in the male subgroup, an earlier NHANES studies also demonstrated higher mean concentrations of PFOS, PFOA and PFHxS in males than in females [17], the significant effect of PFAS and liver enzymes at low doses described above might be a reason to explain the sex differences. Considering the inevitable differences in lifestyle and exposure patterns such as product use, chemical plant work environment, etc. between the genders, further research on this topic is urgently needed.
The four liver enzyme indicators we selected can largely reflect the impairment of liver function [9, 12, 14]. However, each individual index has its own differences in sensitivity and specificity, it is still challenging to reflect the status of liver function with differences in biomarker changes only, and it remains a hot topic whether the possible effects of PFAS are clinically significant compared to other risk factors for liver enzyme effects, specifically: higher ALT is a marker of hepatocyte dysfunction, and as one of the most common one of the liver function tests, with a reference value of less than 40 units, is the main diagnostic item for hepatocellular parenchymal damage, its high level often parallels the severity of the disease and is commonly used to screen children for nonalcoholic fatty liver disease (NAFLD) [62], but 1) there is a lack of consistency between changes in ALT activity and pathological histological changes in the liver, some patients with severe liver damage do not have elevated ALT; 2 ) ALT suffers from the lack of specificity, and there are various reasons that can cause changes in hepatocyte membrane permeability, such as: fatigue, alcohol consumption, colds and even emotional factors; the normal value of AST is 0–37 µ/L, but it is present in hepatocytes and cardiomyocytes at the same time, and even the levels in cardiomyocytes are higher than in hepatocytes, which largely limits its application, and in clinical work we often combine AST with ALT, when ALT is significantly elevated with the ratio (also known as De Ritis ratio) of glutathione (AST)/glutathione (ALT) > 1, it indicates damage to the liver parenchyma and can also be used as an auxiliary test for myocardial infarction and myocarditis [63–65] ; the normal participation value of ALP is 30-90u/L, and the increased level represents impaired biliary excretion in the intrahepatic biliary tract, which is mainly used for obstructive jaundice, primary hepatocellular carcinoma, secondary hepatocellular carcinoma, cholestatic hepatitis, etc. However, this enzyme is also active in bone tissue, and serum ALP can also be elevated in pregnant women, healing fractures, osteochondrosis, osteoporosis, leukemia, and hyperthyroidism; GGT is very low in healthy human serum (less than 40 units), mainly from the liver, with slightly produced by the kidney, pancreas, and small intestine, and GGT is not as good as ALT in reflecting necrotic damage to hepatocytes, and 1) GGT is not as good as ALT in reflecting the necrotic damage of liver cells, but it can be used to distinguish jaundice caused by internal and external liver obstruction, 2) acute and chronic viral hepatitis, cirrhosis, 3) acute and chronic alcoholic hepatitis and drug-related hepatitis: GGT can be significantly or moderately elevated (300–1000 U/L), while ALT and AST are only mildly elevated or even normal, 4) alcoholics can have their GGT decreased after they stop drinking, 5) GGT can also be elevated in other toxic liver disease, fatty liver, liver tumors. Thus, even though we simultaneously found that PFOA exposure resulted in elevated levels of all four liver enzymes and was able to suggest a state of hepatic impairment as assessed by quality of evidence as well as bias analysis, it is still challenging to point specifically to a specific hepatic impairment disease.
Pending exploration of whether such exposure to long-chain compounds (e.g., 9-carbon PFNA) may increase liver enzyme levels is particularly important for the implementation of more stringent regulation of long-chain PFAS [66, 67], and future studies should pay attention to the effects of long-chain PFAS as well as the potential mechanisms [68, 69].
Notably, the results of the meta-analysis under the random effects model and the quality effects model were inconsistent, cutting all the statistically significant combinations. The conflicting results suggest that the need for further research to expanded number of studies and determine the relationship between each exposure and outcome.