The prevalence of ALT elevation unexplained by viral hepatitis, hemochromatosis, or alcoholism (i.e., suspected NAFLD) was 10.6% in NHANES 2003–2004, which was nearly double the prevalence (5.4%) reported by a study of NHANES 1988–1994 adult participants that used similar exclusion criteria and a similar ALT reference range (Clark et al. 2003). As in our study, Clark et al. (2003) also noted that ALT elevation was associated with BMI, Hispanic ethnicity, and middle age. The observed increase in the prevalence of ALT elevation from NHANES 1988–1994 to NHANES 2003–2004 is consistent with the growing burden of obesity and NAFLD.
Because liver biopsy was not performed in NHANES, we used unexplained ALT elevation as a proxy measure of liver disease and NAFLD and identified several ubiquitous environmental pollutants that were dose-dependently associated with suspected NAFLD, including lead, mercury, and PCBs. Although levels of many pollutants are decreasing in the environment, PCB, lead, and mercury exposures remain problematic. For example, even though PCBs were banned in 1977, 100% of subjects in this study had detectable PCB levels.
Diet-induced obesity probably plays the primary role in the pathogenesis of most cases of NAFLD (Cave et al. 2007), but nutrient–toxicant interactions and genetic susceptibility to environmental pollution may be important cofactors, which we did not address in this study. Data from our group and others suggest that diet-induced obesity and fatty liver decrease antioxidant defenses and impair xenobiotic metabolism and disposition, which could sensitize the liver to chemical injury (Fisher et al. 2009a, 2009b; Kirpich et al. in press). Further complicating this issue, lead, mercury, and coplanar PCBs concentrate within the liver, whereas non–dioxin-like PCBs concentrate in adipose tissue and possibly in steatotic (fatty) livers [Klein et al. 1972; Mudipalli 2007; National Toxicology Program (NTP) 2006a]. Therefore, tissue levels may not always correlate with serum levels. However, it is important to recognize that multiple animal studies demonstrate that PCBs and methylmercury (MeHg) exposures induce fatty liver, even in the absence of diet-induced obesity (Chang and Yamaguchi 1974; Desnoyers and Chang 1975b; Lin et al. 1996; NTP 2006a, 2006b, 2006c). Although lead has been associated with hepatic hyperplasia and not NAFLD, to our knowledge lead and diet-induced obesity coexposures have not been performed in animal models (Mudipalli 2007). The results of these aforementioned studies lend biologic plausibility to the hypothesis that lead, mercury, and PCBs may play a previously unsuspected role in the pathogenesis of some cases of suspected NAFLD.
PCBs are polyhalogenated aromatic hydrocarbons that consist of up to 10 chlorine atoms attached to a biphenyl group. About 130 of the 209 theoretical PCB congeners were manufactured between 1929 and 1977 as mixtures and were sold as a function of chlorine content. For example, Monsanto marketed Aroclors 1221, 1231, and 1242 up to 1268, which contain, respectively, 21%, 31%, and 42% to 68% chlorine by weight. Aroclors were used in multiple industrial applications and were components in dielectric insulating fluids for transformers and capacitors, hydraulic fluids, plastics, and paints. An estimated 1.3 million tons of PCBs were manufactured almost exclusively (97%) in the northern hemisphere (Breivik et al. 2002). Although PCBs have been banned in the United States for > 30 years, their high thermodynamic stability makes them resistant to biodegradation and thus persistent organic pollutants. More highly chlorinated PCBs tend to be metabolized and eliminated more slowly, and the PCBs identified in biologic samples in this study were indeed the more highly chlorinated varieties.
From a mechanistic standpoint, a PCB’s structure determines its ability to interact with nuclear receptors. Like PCDDs and PCDFs, coplanar PCBs are aryl hydrocarbon receptor (AhR) agonists, and PCB-126 accounts for 52% of the toxic equivalency of dioxin-like PCBs in human tissues (NTP 2006b; Safe 1993). In comparison, some non–dioxin-like PCBs such as PCB-153 do not activate AhR but may be constitutive androstane receptor agonists (Dean et al. 2002). Animal studies demonstrate that non–dioxin-like PCBs such as PCB-153 are concentrated most heavily within the adipose tissue because of their high lipid solubility (NTP 2006b). Coplanar PCBs, such as PCB 126, despite high lipid solubility, paradoxically concentrate primarily within the liver (NTP 2006b). In our study, both types of PCBs, including PCB-126 and PCB-153, were dose-dependently associated with ALT elevation.
Extensive animal studies conducted by the NTP and others have defined a role for PCBs in liver disease. The NTP has performed 2-year toxicity studies on PCB-126 and PCB-153 in female Harlan Sprague-Dawley rats (NTP 2006a, 2006b, 2006c). These studies demonstrated that the liver was the principal target organ for these compounds. Both benign (toxic hepatopathy, including steatosis) and malignant (hepatocellular carcinoma and cholangiocarcinoma) liver lesions were observed at high frequencies in a dose-dependent fashion, particularly in animals treated with PCB-126 alone or combined with PCB-153. Importantly, both of these PCBs were associated with human ALT elevation in our study. Hennig et al. (2005) demonstrated that PCB-77 exacerbated high-fat-diet (corn oil)–induced hepatic steatosis in mice and increased hepatic gene expression of genes involved in apoptosis, inflammation, and oxidative stress. However, this particular coplanar PCB was not measured in NHANES 2003–2004. In contrast to animal studies, human data on PCBs in liver disease are lacking. However, in Taiwan, 13 years after the “Yucheng” incident where cooking oil was contaminated by PCBs, the mortality rate due to cirrhosis was 2.7 times higher than expected (Yu et al. 1997).
Whole-blood total mercury, present in 92.5% of subjects, but not urinary total (inorganic plus elemental) mercury, was dose-dependently associated with ALT elevation and suspected NAFLD. These results suggest that the organic form of mercury was associated with liver disease. MeHg is the principal form of organic mercury historically associated with organ toxicity. Since the 1950s outbreak of Minamata disease (MeHg intoxication) in a Japanese fishing village, MeHg has been recognized as one of the most hazardous environmental pollutants. Coal-fired power plants have been identified as the primary source of current mercury emissions, and atmospheric mercury may be converted into MeHg in water-body sediment and subsequently enter the aquatic food chain and bioaccumulate in fish (Charnley 2006). The primary route of human MeHg exposure is consumption of contaminated fish and shellfish, and PCB coexposure may occur (Charnley 2006). MeHg has well-characterized toxic effects on the human nervous system, developing fetus, and kidney (Charnley 2006).
Despite the fact that MeHg concentrates considerably within the liver because of enterohepatic recirculation, few animal studies have examined the potential role of MeHg in liver disease. However, acute and chronic toxicity studies conducted in rats and cats demonstrated that mercury exposure resulted in the depletion of body fat, the development of centrilobular hepatic steatosis, an increase in lipid peroxidation products, the proliferation of the endoplasmic reticulum, and floccular degeneration of the mitochondria with extrusion of diseased organelles into the sinusoidal space (Chang and Yamaguchi 1974; Desnoyers and Chang 1975a, 1975b; Klein et al. 1972; Lin et al. 1996). Many of these changes were irreversible after exposure to MeHg was discontinued. The primary mechanism of MeHg hepatotoxicity may be related to its high affinity for sulfhydryl residues and consequent poisoning of cysteine-containing proteins and glutathione depletion (Lin et al. 1996). Previous human epidemiological studies have inconsistently linked mercury contamination in Japanese fishing villages to increased liver-related mortality in villagers (Futatsuka et al. 1987, 1992, 2005).
With a detection rate of 99.6%, lead exposure was nearly universal in adult NHANES subjects. In contrast to PCBs and MeHg, lead hepatotoxicity is relatively well recognized and was recently reviewed (Mudipalli 2007). Lead exposure most commonly occurs through the respiratory or gastrointestinal system. Regardless of the route of exposure, the liver is the largest lead repository in the body (Mudipalli 2007). The pathologic liver lesion of lead exposure has been termed “lead-induced hepatic hyperplasia,” but hepatic steatosis has not been reported. Multiple molecular events have been described in association with lead-induced hepatic hyperplasia. Oxidative stress, proinflammatory cytokine production and sensitivity, and liver and serum cholesterol levels were all increased by lead (Aykin-Burns et al. 2003; Honchel et al. 1991; Kojima et al. 2004; Milosevic and Maier 2000; Sandhir and Gill 1995).
Several potential problems are inherent to the design of this study. The exact specificity of ALT for liver disease in NHANES is unknown because liver biopsies were not performed. However, ALT should be relatively specific, because the incidence of myopathy, the most important extrahepatic source of ALT, is likely low in the general population (Green and Flamm 2002). In contrast, at the reference range used in this study, the sensitivity of ALT is likely lower than its specificity. In fact, some authors have suggested using lower laboratory cutoffs to gain more sensitivity (Prati et al. 2002; Ruhl and Everhart 2009). Importantly, ALT may be normal in NAFLD, and this appears to be an even bigger problem in fatty liver and TASH due to some industrial chemicals (Brautbar and Williams 2002; Cave et al. 2010). Therefore, low-level environmental pollution may pose an even greater risk for liver disease in the general U.S. population than suggested by the results of this study. Lastly, the cross-sectional study design of NHANES cannot determine the direction of causation for the identified associations between environmental pollutant levels and elevated ALT. It is possible that these pollutant concentrations may be elevated because of the presence of liver disease or another predisposing factor for elevated ALT, rather than the risk of elevated ALT being increased because of elevated pollutant levels.
The pollutant subclassifications created by NHANES, although generally reasonable, may not always have the most biologic relevance. For example, heavy metals were grouped differently according to the method of measurement (blood or urine). Given the large number of measured pollutants, looking at all possible groupings of pollutants and mixtures of subgroups was not practical. However, we created new PCB subclasses for coplanar and total PCBs because these molecules were consistently associated with ALT elevation.
Regarding PCBs, NHANES reported levels for only a quarter of the 130 manufactured PCB congeners, so it must be acknowledged that this study did not actually model the effects of total lipid-adjusted serum PCB burden. However, because PCBs were sold in mixtures, it is likely that subjects high in the measured PCBs would also be high in the others. As with all other subclasses, members of the tetrachlorodibenzo-p-dioxin, PCDDs and coplanar PCB subclasses were ranked by serum concentration, which did not account for their toxic equivalency factors. This method allowed us to combine the coplanar PCB and non–dioxin-like PCB subclasses to form the total PCB subclass. However, AhR-dependent hepatotoxicities could be examined by alternate models. Also, although ranking individuals on the basis of exposure levels rather than modeling serum pollutant levels directly allowed us to compare results between individual pollutants and pollutant subclasses, this approach limits comparisons with other study populations.
PCBs, lead, and mercury are present in nearly all U.S. adults. These common pollutants are associated with significant dose-dependent increased ORs for ALT elevation in subjects whose ALT elevations were not explained by viral hepatitis, hemochromatosis, or alcohol abuse. These results suggest a possible association between low-level environmental pollution and the development of liver disease and suspected NAFLD. Future studies should be performed to confirm the potential role of these environmental pollutants in NAFLD.