Abstract
In the U.S., Hepatocellular carcinoma (HCC) incidence rates have increased. We aimed to determine whether environmental exposure plays a role in the high incidence of HCC observed in New York City. We conducted a hospital-based case only study to examine the prevalence of aflatoxin B1 (AFB1)- and polycyclic aromatic hydrocarbon (PAH)-albumin adducts and the distribution of adducts by different characteristics of HCC patients. Blood samples were collected from 155 HCC patients for biomarker analyses. We observed that about 46% and 49% of cases had detectable AFB1- and PAH-albumin adducts, respectively. There were significant differences between AFB1-albumin adducts and selected factors such as HCV infection status (p = 0.04), diabetes (p = 0.03) and Barcelona Clinic Liver Cancer stage (p = 0.02). Cases with detectable PAH-albumin adducts had a smoking history compared with those with nondetectable levels (p = 0.04). The level of AFB1-albumin adducts was positively correlated with plasma bilirubin (rs = 0.32, p < 0.0001) and adiponectin concentrations (rs = 0.28, p = 0.0005). The level of aflatoxin B1-albumin adducts was negatively associated with blood albumin concentration (rs = − 0.28, p = 0.0009) and plasma DNA LINE-1 methylation (rs = − 0.16, p = 0.04). Our study provides additional evidence that environmental exposures including to aflatoxin might drive the high incidence of HCC observed in the New York City.
Keywords: Aflatoxin, Aflatoxin B1-albumin adduct, Hepatocellular carcinoma, LINE-1 methylation, Adiponectin, Polycyclic aromatic hydrocarbon
Introduction
Hepatocellular carcinoma (HCC) death rates are rising, even as most other cancers in the US are declining [1]. In New York City (NYC), HCC is one of the leading causes of cancer-related death. The risk of dying from HCC is much greater for people who live in the Bronx and nearby neighborhoods that are predominantly populated by Blacks and Hispanics, compared to other parts of NYC and the US [2]. Differences in traditional risk factors likely contribute to HCC disparity [2], but less is known about the role of environmental exposure in both geographic and racial disparities.
Aflatoxins, genotoxic hepatocarcinogens, are toxic fungal metabolites that commonly contaminate crops. Polycyclic aromatic hydrocarbons (PAHs), possible human carcinogens, are ubiquitous pollutants caused by incomplete combustion of various materials, including diesel fuel and tobacco [3]. Using a prospective study design, we reported that higher levels of aflatoxin B1- (AFB1) and PAH-albumin adducts were associated with HCC risk in Taiwan where the incidence of HCC is high [4]. A recent survey of 184 volunteers recruited in a Hispanic community in South Texas where the incidence of HCC is high reported aflatoxin adducts were detected in 21% of the participants with a mean level fivefold higher than that detected in the US general population [5]. These observation suggest that aflatoxin might explain some of the epidemiology of US HCC in term of the significant geographic and ethnic variations in incidence [1].
Here, we conducted a hospital-based case only study to examine the prevalence of AFB1- and PAH-albumin adducts and the distribution of adducts by different characteristics of HCC patients that were treated in a medical center located in the heart of the Washington Heights neighborhood of northern Manhattan.
Materials and methods
Patients
This study used information from a total of 155 adult patients (≥ 18 years old) with newly diagnosed HCC and treated in the Hepatobiliary Oncology Clinics at the Columbia University Irving Medical Center (CUIMC) during October 2008 to July 2014. Subjects completed an epidemiologic questionnaire, underwent a physical examination (including weight and height measurements) and provided a fasting morning blood sample at the time of enrollment. Information on biochemical blood analyses, including viral hepatitis, albumin, and total bilirubin, were obtained from the medical records. Simultaneously, an additional 30 mL of blood was processed to isolate plasma and buffy coat which were frozen at − 80 °C. This study was approved by the CUIMC Institutional Review Board.
Aflatoxin B1- and PAH-albumin adduct measurement
We used ELISA assay for aflatoxin-and PAH-albumin adducts [4]. Briefly, 50 μl of albumin extracts, equivalent to 200 μg of albumin, were added to 96-well plates (Easy-wash, Corning, NY) previously coated with 5 ng of 7β, 8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo(a) pyrene (BPDE-I)-DNA. The standard curve was generated by adding serial dilutions of BPDE-I-tetrols in PBS from 5 to 2,500 fmol in 50 μl into the coated wells followed by 50 μl of antibody. The values for each subject were expressed as fmol of PAH per mg albumin. Samples with less than 15% inhibition were considered undetectable and assigned a value of 50 fmol/mg albumin.
For AFB1-albumin adduct were assayed by ELISA: enzyme digested albumin was extracted and the equivalent of 200 μg of albumin added to 96-well plates previously coated with 3 ng AFB1 epoxided-modified human serum albumin. Polyclonal antiserum seven was used and samples with < 20% inhibition were considered non-detectable and assigned a value of 4.5 fmol/mg.
Measurement of LINE-1 methylation
Plasma DNAs were isolated using QIAmp UltraSense Virus Kits (Qiagen, Valencia, CA). Aliquots of DNA (500 ng) were bisulfite-treated with the EZ DNA methylation kit (Zymo Research, Orange, CA) following the manufacturer’s protocol. Pyrosequencing for LINE-1 methylation levels was carried out using PCR and sequencing primers as described previously [5]. Briefly, PCR was carried out in a 25 μl reaction mix containing 50 ng bisulfite-converted DNA, 1X Pyromark PCR Master Mix (Qiagen), 1X Coral Load Concentrate (Qiagen) and 0.2 μM forward and reverse primers. Following amplification, the biotinylated PCR products were purified and incubated with the sequencing primer designed to bind adjacent to the CpG sites of interest. Pyrosequencing was conducted using a PyroMark Q96 instrument (Qiagen), with subsequent quantitation of methylation levels determined with the PyroMark CpG 1.010 software. Relative peak height differences were used to calculate the percentage of methylated cytosines at each given site. Percent methylation within a sample was subsequently determined by averaging across all three interrogated CpG sites in the analysis.
Plasma adiponectin measurement
Human adiponectin levels was determined in duplicate using commercial ELISA Kits (ThermoFisher Scientific, Camarillo, CA, USA). A serial dilution of adiponectin (from 0.5 to 32 ng/ml) was used to generate a standard curve.
Statistical analysis
We used the χ2 test for categorical variables and Student’s t-test for continuous variables to assess the difference in selected characteristics between exposure groups (detectable vs undetectable adducts). We calculated the Spearman rank correlation coefficients (rs) to determine the correlation of adducts and plasma LINE-1 methylation. All analyses were performed with SAS software 9.4 (SAS Institute, Cary, NC).
Results
The racial distribution of our HCC cases is diverse and includes 48% whites, 25% of Hispanics, 13% Asian and 10% Blacks. There were about 46% and 49% of cases with detectable AFB1- and PAH-albumin adducts, respectively. The mean level was 10.8 fmol/mg for AFB1-albumin adducts and 116.9 fmol/mg for PAH-albumin adducts.
There were significant differences between AFB1-albumin adducts and selected factors such as HCV infection status (p = 0.04), diabetes (p = 0.03) and Barcelona Clinic Liver Cancer (BCLC) stage (p = 0.02) (Table 1). Cases with detectable PAH-albumin adducts were older (p = 0.02) and had smoking history compared with those with nondetectable levels (p = 0.04).
Table 1.
The distribution of aflatoxin B1 (AFB1)- and polycyclic aromatic hydrocarbon (PAH)-albumin adducts by selected characteristics among hepatocellular carcinoma cases
| n (%) | AFB1-albumin adducts | p value | PAH-albumin adducts | p value | ||
|---|---|---|---|---|---|---|
| Detectable | Undetectable | Detectable | Undetectable | |||
| n = 71 (46%) | n = 84 (54%) | n = 73 (49%) | n = 76 (51%) | |||
| Age, mean (SD) Gender | 61.5 (10.8) | 64.3 (9.8) | 0.11 | 65.4 (10.6) | 61.7 (8.3) | 0.02 |
| Female | 18 (26) | 16 (19) | 0.34 | 14 (19) | 19 (25) | 0.39 |
| Male | 53 (74) | 68 (81) | 59 (81) | 58 (75) | ||
| Race | ||||||
| White | 38 (53) | 37 (46) | 0.1 | 41 (56) | 30 (39) | 0.01 |
| Hispanics | 14 (20) | 25 (30) | 20 (27) | 18 (24) | ||
| Blacks | 11 (15) | 5 (6) | 4 (5) | 11 (15) | ||
| Asian | 6 (8) | 14 (17) | 7 (10) | 13 (17) | ||
| Other | 2 (2) | 3 (4) | 1 (1) | 4 (5) | ||
| Foreign born | ||||||
| No | 38 (56) | 41 (55) | 0.63 | 41 (58) | 36 (51) | 0.45 |
| Yes | 30 (44) | 34 (45) | 30 (42) | 34 (49) | ||
| Education | ||||||
| Less than high school | 34 (50) | 31 (39) | 0.19 | 29 (41) | 34 (49) | 0.36 |
| High school and above | 34 (50) | 48 (61) | 42 (59) | 36 (51) | ||
| Annual income | ||||||
| < $30,000 | 21 (37) | 26 (39) | 0.78 | 23 (39) | 23 (38) | 0.71 |
| $30,000–60,000 | 14 (25) | 13 (19) | 15 (25) | 12 (20) | ||
| > $60,000 | 22 (39) | 28 (42) | 21 (36) | 25 (42) | ||
| HCV | ||||||
| Negative | 24 (34) | 42 (50) | 0.04 | 35 (48) | 30 (39) | 0.3 |
| Positive | 47 (66) | 42 (50) | 38 (52) | 46 (61) | ||
| HBV | ||||||
| Negative | 62 (87) | 65 (77) | 0.11 | 63 (86) | 58 (76) | 0.12 |
| Positive | 9 (13) | 19 (23) | 10 (14) | 18 (24) | ||
| Alcohol use | ||||||
| Never | 27 (38) | 29 (34) | 0.65 | 23 (31) | 31 (41) | 0.24 |
| Ever | 44 (62) | 55 (66) | 50 (69) | 45 (59) | ||
| Smoking history | ||||||
| Never | 22 (32) | 36 (46) | 0.1 | 23 (33) | 35 (49) | 0.04 |
| Ever | 46 (68) | 43 (54) | 47 (67) | 36 (51) | ||
| Diabetes | ||||||
| No | 47 (66) | 41 (49) | 0.03 | 38 (52) | 48 (63) | 0.17 |
| Yes | 24 (34) | 43 (51) | 35 (48) | 28 (37) | ||
| BCLC stage | ||||||
| 0 or A | 20 (29) | 38 (46) | 0.02 | 29 (40) | 28 (37) | 0.76 |
| B or C | 50 (71) | 45 (54) | 44 (60) | 47 (63) | ||
p values < 0.05 are bolded
Figure 1 presents the statistically significant correlation of AFB1-albumin adducts and selected blood markers. There was a positive association between AFB1-albumin adducts and total bilirubin concentration, with a correlation coefficient of 0.32. The level of AFB1-albumin adducts was negatively associated with blood albumin concentration (rs = − 0.28, p = 0.0009) and plasma DNA LINE-1 methylation (rs = − 0.16, p = 0.04). The correlation coefficient between AFB1-albumin adducts and plasma adiponectin concentration was 0.28 (p = 0.0005). No associations were found between PAH-albumin adducts and blood markers.
Fig. 1.
The correlation of aflatoxin B1-albumin adducts and blood markers. A The correlation of aflatoxin B1-albumin adducts and total bilirubin concentration (rs = 0.32, p < 0.0001). B The correlation of aflatoxin B1-albumin adducts and blood albumin concentration (rs = − 0.28, p = 0.0009). C The correlation of aflatoxin B1-albumin adducts and plasma DNA LINE-1 methylation (rs = − 0.16, p = 0.04). D The correlation of aflatoxin B1-albumin adducts and plasma adiponectin concentration (rs = 0.28, p = 0.0005)
Discussion
Our results indicate that both aflatoxin and PAH exposures are common in US HCC cases. Although it is estimated that 0.7% of US HCC show evidence of aflatoxin exposure from the Cancer Genome Atlas analysis [6], we observed that ~ 46% of HCC cases had detectable aflatoxin adducts. Moreover, we observed about 69% of cases with at least one type of adducts: 23% of cases with both AFB1- and PAH-albumin adducts, 26% of cases with only PAH-albumin adducts, and 20% of cases with only AFB1-albumin adducts. We did not observe differences in adduct levels related to year of HCC diagnosis (data not shown). Our study provides additional evidence that environmental exposure including to aflatoxin might drive the high incidence of HCC observed in the New York City.
Aflatoxin contamination in foods has become a global problem because of climate change [7]. Aflatoxin contamination of crops is difficult to combat because it can contaminate important food crops such as spices during the growing seasons and after harvest during storage [7, 8]. Many technologies such as ELISA and high-performance liquid chromatography (HPLC) have been developed to measure aflatoxin albumin adducts as a biomarker for the assessment of chronic exposure to AFB1 in many epidemiological studies assessing the health effects related to exposure [9]. AFB1-albumin adduct concentrations measured by ELISA are well correlated with AFB1-lysine concentrations measured by HPLC and mass spectrometry [10, 11]. A survey of 184 volunteers recruited between 2007 and 2008 in a Hispanic community in South Texas where the incidence of HCC is high reported aflatoxin-albumin adducts were detected in 21% of the participants with a mean level fivefold higher than that detected in the general US population using National Health and Nutrition Examination Survey 1999–2000 [5, 12]. An increase in aflatoxin adducts over time was also reported in another survey using bloods from volunteers recruited from 2004 to 2014 [13]. In our un-published data, we measured AFB1-albumin adducts in 65 health volunteers recruited within CUIMC. We found the mean AFB1-adducts was similar to the mean reported in a 2014 survey at Texas (3.4 pg/mg at CUIMC vs 3.98 in Texas) [13]. While some of these differences in absolute adduct levels may be related to the different assays used in the different studies, our data suggest that hepatocarcinogens are present at high levels in HCC cases in a community with a high burden of HCC. Unfortunately, we did not collect dietary questionnaire data from our participants and are not able to explore the correlation of aflatoxin exposure to specific dietary factors. In a prior study, the amount of rice, corn tortillas and nuts consumed was found to be significantly associated with urinary aflatoxin metabolites [5].
Aflatoxin, a genotoxic hepatocarcinogen, can induce G → T mutations, in particular at codon 249 p53 [14]. A significant racial difference in P53R249S mutations, a hallmark of aflatoxin-related HCC, in tumors from HCC cases in South Texas was also observed; The prevalence of TP53R249S mutations was highest in Asians (72.7%), followed by Blacks (18.2%), and Hispanics (9.1%) [15]. In our study, we did not see any racial difference in AFB1-albumin adducts. However, measuring AFB1-DNA adducts and p53 mutations in HCC tumors, we previously observed that AFB1-DNA adducts were associated with p53 DNA mutation [14]. As adducts serve as a biomarker of exposure, examining the mutational spectrum of tumors might assist in the better understanding of racial differences in mechanisms of AFB1 carcinogenesis.
A strong interaction between aflatoxin and HBV was reported in Asia where the prevalence of HBV was high [16, 17]. In our case–control study nested in a community-based cohort in Taiwan, we found that HCC risks was six-fold higher for individuals with chronic HCV infection and low AFB1-albumin adduct, and 20-fold for individuals with both chronic HCV infection and high adducts compared to those with low adducts and no HCV infection [18]. In the present study, we observed that individuals with detectable AFB1-albumin adduct were more likely to be HCV positive. Further study is needed to better understand the molecular mechanism involved in AFB1 promoting hepatocarcino-genesis in HCV-infected liver. We did not observe a difference of AFB1-albumin adducts by HBV infection status. This might be related to small number of HCC participants positive for HBV infection. We observed that about 34% of HCC cases with detectable of AFB1-albumin adducts had diabetes, while 51% of cases without detectable of AFB1-albumin adducts had diabetes. Without a control group, we cannot conclude that patients with diabetes are less likely to develop HCC. Cases with diabetes might be more likely to eat fresh food and thus, have less AFB1 exposure compared with cases without diabetes.
We found AFB1-albumin adducts were associated with blood markers including total bilirubin, albumin, plasma LINE-1 methylation and adiponectin. Adiponectin is known to regulate inflammation, tumor microenvironment and behavior [19]. A study of HCC tissues found that increased adiponectin significantly correlated with tumor size, indicating its possible role in prediction of poor prognosis [20]. We previously reported lower plasma levels of LINE-1 methylation was associated with worse survival in HCC patients [21]. Both animal and human studies found aflatoxin is immunosuppressive by impairing and suppressing normal immune function [22]. Further study is need to understand the potential effects of aflatoxin exposure on HCC prognosis.
Analyzing plasma banked up to 12 years before diagnosis, we demonstrated a twofold increased risk for development of HCC with increased PAH-albumin adducts independent of viral status [4]. In the present study, we found that about half of the HCC cases had detectable PAH-albumin adducts and these with adducts were more likely to be ever smokers. Previous studies have suggested a role for smoking in development of HCC [23]. That the correlation of PAH-albumin adduct and smoking suggest that PAH-albumin adducts can be used a biomarker for validating smoking cessation in HCC prevention.
Study strengths include using biomarker measurement to quantify environmental exposure at the individual level. The limitations of this study are small sample size and case only study design. A larger study will be needed to identify individuals at high risk of aflatoxin exposure which will useful for HCC prevention.
Acknowledgments
This work was supported by awards from the National Cancer Institute [R01ES005116 and P30 CA013696] and the National Institute of Environmental Health Sciences [P30 ES009089]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Footnotes
Conflict of interest The authors report no conflict of interest.
Data sharing Contact the corresponding author for any inquiries regarding data or analytical code.
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