Serum concentration of polychlorinated biphenyls and risk of hepatocellular carcinoma: a case-control study in a highly polluted area in Italy

Background: Polychlorinated biphenyls (PCBs) are human carcinogens, based on sucient evidence for melanoma and limited evidence for non-Hodgkin lymphoma and breast cancer. Few data are available for liver cancer, although PCBs cause it in rats and determined liver damage in poisoned people. We investigated the association between PCB serum levels and hepatocellular carcinoma (HCC) with a case-control study in a PCB-polluted area in North Italy. Methods: We enrolled prospectively 102 HCC incident cases and 102 age- and gender-matched hospital controls. Serum concentrations of 33 PCB congeners were determined by a gas chromatograph coupled to mass spectrometry. Results: Of the 102 HCC cases, 62 who had lost < 3 kg of body weight in past 3 years were included in the analysis (67.7% males, mean age 68 years). 69% of them (8% of controls) had one or more risk factors for HCC: past alcohol intake ≥ 60 g/day and/or HBV or HCV infection. The odds ratio (OR) for HCC for 3 rd compared to 1 st tertile of PCB distribution was 1.76 (95% condence interval 0.62-5.03) for total PCB, adjusting for socio-demographic variables and risk factors for HCC by logistic regression. No differences were found restricting the analysis to HCC cases and controls with or without the main risk factors for HCC. For most PCB congeners, ORs > 1.5 or 2 were found, although the 95% CIs included the null value for almost all of them. Conclusions: This preliminary study suggests that PCBs might play a role in HCC development.

was found in a cross-sectional study on a residential US cohort with elevated PCB exposure (4), and an association between PCB serum levels and unexplained ALT elevation was observed in a multicentre US cross-sectional survey (5). PCB exposure causes liver cancer in rats, but occupational cohort studies showed inconsistent results as regards liver cancer mortality (6), and no epidemiological study on the association between PCBs and HCC using biological measures of PCB exposure has been conducted so far, to our knowledge.
A chemical factory located in Brescia, the main town in the province, produced PCBs and other organochlorines from the 1930s to 1984 determining a heavy contamination of the soil, surface water sediments and locally produced food, including animal products (milk and cheese, meat, chicken and eggs) (7,8,9). A study on a random sample of adults living in the town showed higher PCB serum concentrations than those usually found in people living in industrial area and not occupationally exposed, up to about 40,000 ng/g of lipid-adjusted serum total PCBs (10).
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for more than 80% of primary liver cancers worldwide. A high incidence of HCC has been registered in the Brescia area as compared to others in Italy (11,12). A case-control study carried out in 1995-2001 on 464 cases and 824 controls showed that hepatitis C virus (HCV), hepatitis B virus (HBV) and consumption of more than 60 grams of ethanol per day for at least one decade were responsible for about 90% of the total cases occurring in the area (13,14), in agreement with ndings from other countries (15). Also a role of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) in HCC development has been demonstrated recently (16,17), but this condition seems still uncommon in our country, being responsible for about 5% of the total HCC cases in a recent large multicentre Italian study (18). However, a role of other factors, including environmental toxics for HCC development, cannot be excluded. We carried a preliminary case-control study aimed to investigate the possible association between PCB exposure and HCC, taking account of the major risk factors for HCC in Western countries.

Study population
We carried out a hospital-based case-control study. The study base was the population living in the province of Brescia and born in Italy. The cases were recruited consecutively and prospectively in two Divisions (General Surgery and Hepatology) of the main General Hospital of the province. 96% of the eligible cases agreed to participate in the study.
Overall, we enrolled 102 patients with a rst diagnosis of HCC (incident cases) in 2015-2018, before they underwent any treatment for the disease. The diagnosis of HCC was based on computerised tomography and was con rmed by histological analyses in 63 cases.
We selected 102 control subjects in the same age range and gender as the cases and without history of cancer or hepatic, endocrine or autoimmune diseases, who were admitted to the same hospital at the same time as the cases.
When a subject suitable for the study according to the inclusion criteria was identi ed, he/she was invited to participate, and, in case of refusal, another individual was chosen and invited to participate. Overall, about 70% of the eligible subjects participated in the study. The enrolled controls were hospitalized for traumatic causes, vascular surgery or other minor surgery.
Both cases and controls provided a fasting blood sample for laboratory determination and were interviewed at the hospital on demographic variables, weight and height, residential and occupational history, smoking habit and alcohol intake by the research personnel. Since a rapid weight loss may determine an increase of PCB serum levels (19,20), the HCC cases were also questioned about the quantity of, and time at, weight loss in the past.
Total alcohol intake was computed according to the average ethanol content of wine (12 percent by volume), beer (5 percent) and spirits (40 percent) and the frequency and quantity of alcoholic beverages consumed in the past. We considered the intake claimed by the subject during the decade in his/her lifetime with the highest consumption ("peak"), which had provided valuable results for evaluating the dose-effect relationship between alcohol intake and HCC occurrence in previous case-control studies carried out in this area (21). Heavy alcohol intake was de ned as consumption of 60 or more grams of ethanol per day for at least 10 years.
The Ethics Committees of the main hospitals in the area and of the Local Health Authority of Brescia approved the project, and each participant provided a written informed consent.
The PCB analysis was conducted following a previously de ned analytical method (23), using an Agilent Technologies 6890N gas chromatograph coupled with an Agilent Technologies MSD 5973 (electron impact ionization, mass lter: quadrupole). A PONA column (Agilent Technologies; 50m x 0.20 mm ID) was used for chromatographic separation with carrier gas Helium. A 2 mL injection at 250 °C was performed by a 7683 Series Injector (Agilent Technologies) in splitless mode with a salinized injection liner (Agilent Technologies; 4mm, 78.5 x 6.5 OD).
The limit of quanti cation (10 times the signal-to-noise ratio peaks) varied among PCBs but was generally less than 0.1 ng/ml for each congener. Total PCB serum concentration was calculated as the sum of the 33 PCB congeners. Since PCB concentration is in uenced by the amount of serum lipids, the ratio of PCB concentration to the total lipid levels was computed (lipidadjusted PCB concentration) and expressed as ng/g lipid. We calculated the total lipid concentration from cholesterol and triglyceride levels using the formula proposed by Phillips et al. (24): total serum lipid (g/L) = 2.27 * serum cholesterol (g/L) + triglycerides (g/L) + 0.623.
We also investigated the three main risk factors for HCC in this area, i.e. HBV and HCV infection and alcohol intake (10), as potential risk modi ers of the possible association between PCB exposure and HCC. The presence of HBV and HCV infections was evaluated by testing sera for HBsAg and anti-HCV, respectively, using commercial immunoassays (EIA).

Statistical analysis
The distributions of total PCBs and each congener serum levels were examined using common statistical techniques for exploratory analysis. Due to the asymmetric non-normal distribution of PCB values, the median, range and 90 th percentile are reported. The differences in PCB concentration between HCC cases and controls were evaluated using non-parametric methods, particularly the Mann-Whitney test for impaired data. The odds ratios (ORs) for HCC were calculated using the tertiles of serum PCB concentrations in the controls as the cut-offs and adjusting for sex, age, residence, education and presence of one or more risk factors for HCC using multiple logistic regression. The con dence intervals were computed at the 95% level. Furthermore, a test for linear trend was performed using the Wald test on the coe cients based on PCBs as continuous variables.
All the statistical tests were two-sided with a threshold of p=0.05 for refusing the null hypothesis. The statistical analyses were performed using the STATA software for personal computer (Stata Statistical Software release 14.0; Stata Corporation, College Station, Texas).

Results
A total of 102 cases and 102 controls were enrolled. Among HCC cases, 40 said they had lost 3 kg and over of their body weight in the last 3 years. A comparison between HCC cases with and without recent weight loss showed that the former had signi cantly higher PCB serum levels than the latter: the medians of total serum PCBs were 1183 and 757 ng/g lipid, respectively (p=0.02 by Mann-Whitney test). Among the 40 HCC cases with recent weight loss, no relationship was found between either quantity of, or time at, weight loss and PCB serum levels. Since HCC cases with recent weight loss may have higher PCB serum levels due to their release from lipid deposits into the blood, we included only the HCC cases without relevant weight lost in the last three years in the analysis.
Gender, age, residence and education and main risk factors for HCC in both cases and controls are shown in Table 1. HBV infection alone + HBV infection and heavy alcohol intake (60+ g/day).
Age and gender distribution were almost identical between cases and controls, whereas some differences were observed according to residence, with 17.7% of cases and 35.3% of controls living in the town, and education, with 21.0% of cases and 36.3% of controls with more than 8 school years. Of the HCC cases, 41.9% had HCV infection, with or without other risk factors, 6.5% had HBV infection, with or without a history of heavy alcohol intake, 21% had a history of heavy alcohol intake alone, and 30.6% had no risk factors. As expected, few controls showed one or more of the main risk factors for liver disease: 4.9% had HCV or HBV infection and 4.9% had a history of heavy alcohol intake. A total of 33 cases (53%) had a clinical history of cirrhosis.
The median, range and 90 th percentile of lipid-adjusted serum concentration of the PCB congeners found in at least 30% of subjects and of the total PCBs in HCC cases and controls are shown in Table 2. No difference was found in total PCB serum levels in HCC cases according to the presence or absence of at least one of the main risk factors for HCC (Table 3). The 43 HCC cases with one or more risk factors had slightly lower values of total PCBs than the cases without risk factors (medians of 745 and 909, respectively), although the difference was not statistically signi cant.

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The distribution of HCC cases and controls according to tertiles of PCB concentration in the controls, and the corresponding odds ratios (ORs) adjusted for age, sex, residence, education and presence of one or more risk factors for HCC by multiple logistic regression are reported in Table 4. Similar results were found when restricting the analysis to subjects with, and subjects without, the main risk factors for liver disease (table 5): increased ORs for 3 rd vs 1 st tertile of PCB distribution were observed for both groups, although the 95% CIs were large due to the small number of subjects in some categories. No differences were found in subgroup analyses by gender, age and residence (in or out the main town of the area) (data not shown).

Discussion
This preliminary study provides some evidence for an association between HCC and serum levels of PCBs, when also taking account of demographic and socio-economic factors and main risk factors for HCC. These results are consistent for almost all the PCB congeners found in at least 30% of subjects. However, since HCC patients undergo liver cancer after a long history of cirrhosis, some concern may be raised whether the liver function impairment or the weight loss due to cancer development determine an increase of PCB serum levels in these patients (25). In fact, we found that HCC cases who had lost more than 3 kg of body weight in the last 3 years, before HCC diagnosis, had higher PCB serum levels than those who had not lost weight, and we argued that higher PCB serum levels in the former were possibly due, at least partly, to mobilization of various chemicals, including PCBs, stored in body fat deposits. For this reason, we decided to exclude HCC cases with recent body loss of more than 3 kg.
Most HCC cases had one or more major risk factors for HCC, namely HCV and HBV infection and alcohol intake >60 g/day for at least 10 years, in agreement with a previous case-control study on HCC aetiology carried out in the same area (13,21). HCC cases with and without the main risk factors for the disease showed similar PCB serum levels, and the ORs for HCC did not vary substantially stratifying subjects according to the presence or absence of at least one risk factor for HCC, suggesting that PCBs may be independent risk factors for HCC development.
The association between HCC and PCB exposure is biologically plausible as the liver is a target organ of these chemicals and an increased incidence of liver cancer is observed after their administration in rats (2). The different PCB congeners, including dioxin-like and non-dioxin-like compounds, have different activities according to their chemical structure. We found that moderate and high chlorinated PCB congeners were detectable in most subjects and were at the highest serum concentration, particularly congeners 138, 153, 170, 180 and 194, in agreement with those most commonly detected in human studies, (2).
However, PCBs usually occur in complex mixtures eliciting both genotoxic and non-genotoxic effects associated with carcinogenesis, tumour promotion and progression: in-vitro assays and experimental animal studies have shown that PCBs may produce oxidative stress and genotoxicity, interact with various receptors, including the aryl hydrocarbon receptor (AhR) and others controlling xenobiotic and steroid hormone metabolism or modulate plasma membraneassociated proteins affecting cell communication, adhesion and migration (2).
In humans, an association between serum levels of PCB congeners and unexplained ALT elevation was found in the US NHANES (5), contrary to our ndings of no relationship between PCB and ALT serum levels in the general population living in this area (26). In the Anniston Survey, however, a cross-sectional study on a residential cohort with elevated PCB exposure, an association was found between serum levels of PCB congeners and those of the cytokeratin 18 biomarker, a more sensitive biomarker than ALT for environmental liver disease (4).
Few epidemiological data are available on the possible association between PCB exposure and HCC at present. The cohort studies of people who ingested rice oil contaminated with PCBs and polychlorinated dibenzofurans (PCDFs) in the Yusho and Yucheng incidents, in Japan in 1968 and in Taiwan in 1979, found an excess mortality from liver cancer and liver cirrhosis among intoxicated subjects in the rst 10-15 years after the poisoning outbreak (3,27). However, almost 90% of the dioxin-like toxic equivalency (TEQ) in human blood of the contaminated people was contributed by PCDFs in both incidents (28), arising doubts on the actual role of PCBs by themselves. On the contrary, although high levels of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were also found in the general population in Brescia, the major contribution to TEQ serum levels was due to PCBs (8). Some, but not all the occupational cohort studies found an increase in liver cancer mortality in PCB exposed workers with respect to national or regional gures (29,6), but none of them had PCB serum measures.
The main strengths of our study are that it was performed in a highly industrialized area with both high incidence of HCC and heavy exposure to PCB and possibly PCDD and PCDF as a consequence of half a century of PCB diffusion in the environment, with particularly high concentrations of these chemicals in soil, food and blood of residents (7,8,10).
This research has some limitations, too. First of all, the limited number of HCC cases enrolled and the relatively high proportion of those excluded from the analysis due to recent weight loss have reduced the a-priori de ned power of detecting an OR of at least 2 for 3 rd compared to 1 st tertile of PCB distribution at the threshold of p=0.05 using two-tail statistical tests. Therefore, our study can be considered as an explorative investigation on the association between HCC and PCB exposure. The use of PCB serum level as a measure of the body burden of these chemicals in HCC patients may be of concern, due to their history of chronic liver disease. PCBs are highly soluble in lipids and are mainly deposited in adipose tissue and transported in the blood by lipids. HCC cases showed lower mean values of total cholesterol and triglycerides (133.5 and 85 mg/dl) than those found in the Italian population aged 65-74 years (204 and 132 mg/dl, respectively) (30). This nding was expected, since low serum values of total cholesterol and triglycerides are usually found in HCC cases, due to advanced cirrhosis and diet followed before or during hospitalization for the liver disease (31). In HCC patients, the partitioning between adipose tissue and serum is close to 1:1 only when both are expressed on a lipid basis. Indeed, in a previous study of 101 HCC incident cases enrolled in the same area, we found high correlations between serum and liver (Spearman r=0.79), serum and fat (r=0.91), and liver and fat (r=0.75) concentrations of single PCB congeners and total PCBs, using lipid-adjusted PCB measures (32). The retrospective study design allows the comparison only of present PCB serum levels between HCC cases and controls, ignoring past exposures to these chemicals. However, PCB serum levels are usually considered a valid measure of PCB body storage, as shown by the high correlations between PCB serum and adipose tissue levels found in our and other studies (32,33,34,35,36).
As regards the selection of subjects, we enrolled both cases and controls by the same study base, i.e. residents in Brescia province, matched by age and gender, admitted to the same General Hospital. We collected blood samples of both groups at the same time and stored them for a short time before performing blinded laboratory analyses. The HCC cases were prospectively recruited among all the incident ones admitted to the General Surgery and Hepatology Divisions of the main hospital in the area, and they had similar characteristics as those enrolled in previous casecontrol studies on HCC etiology carried out in the same area (10,13) and those reported by the Italian Cancer Registries (37) and enrolled in a recent large multicentre Italian series (18). We choose the controls among subjects hospitalized for traumatic causes, vascular surgery or minor surgery diseases. They showed similar PCB serum levels as the general population of the same age living in the town, as found in a recent survey (26). Therefore, we are con dent that no substantial selection bias exists in this study.

Conclusions
This preliminary study provides some, though not conclusive, evidence that PCB exposure may play a role in HCC development, independently of other risk factors.