# The Effect ofCYP, GST, and SULT Polymorphisms and Their Interaction with Smoking on the Risk of Hepatocellular Carcinoma

Authors: Stefania Boccia; Luca Miele; Nikola Panic; Federica Turati; Dario Arzani; Consuelo Cefalo; Rosarita Amore; Milutin Bulajic; Maurizio Pompili; Gianlodovico Rapaccini; Antonio Gasbarrini; Carlo La Vecchia; Antonio Grieco
Journal: BioMed Research International
 (2015)

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## Abstract

Aim. The aim of our study was to assess whether selected single nucleotide polymorphisms of CYP1A1 and 2E1, GSTM1, GSTT1, and SULT1A1 influence susceptibility towards HCC, considering their interaction with cigarette smoking. Methods. We recruited HCC cases and controls among patients admitted to the hospital “Agostino Gemelli,” from January 2005 until July 2010. Odds ratios (OR) of HCC were derived from unconditional multiple logistic regression. Gene-gene and gene-smoking interaction were quantified by computing the attributable proportion (AP) due to biological interaction. Results. The presence of any CYP2E1*5B variant allele (OR: 0.23; 95% CI: 0.06-0.71) and CYP2E1*6 variant allele (OR: 0.08; 95% CI: 0.01–0.33) was inversely related to HCC. There was a borderline increased risk among carriers of combined CYP1A1*2A and SULT1A1 variant alleles (OR: 1.67; 95% CI: 0.97–3.24). A significant biological interaction was observed between GSTT1 and smoking (AP = 0.48; 95% CI: 0.001–0.815), with an OR of 3.13 (95% CI: 1.69–5.82), and borderline significant interaction was observed for SULT1A1 and smoking (AP = 0.36; 95% CI: −0.021–0.747), with an OR of 3.05 (95% CI: 1.73–5.40). Conclusion. CYP2E1*5B and CYP2E1*6 polymorphisms have a favourable effect on the development of HCC, while polymorphisms of GSTT1 and SULT1A1 might play role in increasing the susceptibility among smokers.


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## Body

## 1. Introduction

Hepatocellular carcinoma (HCC) is currently the sixth most common cancer and the third cause of cancer deaths worldwide [1]. Its prognosis remains poor, with a 5-year survival rate less than 20% in Europe [2]. Risk factors for HCC include infection with hepatitis B (HBV) and hepatitis C viruses (HCV), history of diabetes mellitus, nonalcoholic fatty liver disease and cirrhosis, heavy alcohol consumption, and cigarette smoking [3–5]. Coffee consumption appears to have a favorable effect [6]. Further, genetic factors appear to modulate the individual susceptibility as the siblings of HCC individuals are more prone to develop the HCC even in the absence of HBV infection [7]. So far, several single nucleotide polymorphisms (SNPs) have been investigated in association with HCC, with contradictory results [8].As the liver is the main metabolic organ, the SNPs related to genes encoding carcinogen-metabolizing enzymes represent key target candidates for association analyses. Cytochrome P-450 (CYP) is a superfamily of monooxygenases responsible for phase I enzyme reactions, preparing substrates for phase II conjugation reactions, but they can also lead to the metabolic activation of toxic or carcinogenic compounds [9]. The glutathione S-transferases (GSTs) are a gene superfamily coding phase II enzymes that detoxify free radicals in tobacco smoke, products of oxidative stress, and polycyclic aromatic hydrocarbons [10]. Sulfotransferase (SULT) catalyzes sulfonate conjugation to detoxify the procarcinogens to metabolites, which are easily eliminated from the body [11]. Polymorphisms in these genes, their combination, and interaction with environmental factors have the potential to lead to increased susceptibility to HCC. While some studies investigated the effect ofGST andCYP genes on HCC, as well as their combination and interaction with smoking [12, 13], little information is available on the effect ofSULT1A1.The aim of our study was to assess whether the selected SNPs ofCYP1A1 and2E1,GSTM1, GSTT1,andSULT1A1 genes influence individual susceptibility to HCC, also considering their combination and interaction with cigarette smoking.


## 2. Methods

### 2.1. Study Population

Study participants were recruited among patients admitted to the teaching hospital “Agostino Gemelli” of the Università Cattolica del Sacro Cuore (Rome, Italy) from January 2005 until July 2010, and eligibility was restricted to white individuals born in Italy. Cases were 221 patients with HCC recruited among subjects referred to the Outpatient Liver Unit of the hospital. The diagnosis of HCC was performed according the AASLD guidelines [14]. The control group included 290 patients from the same hospital with a broad range of diagnoses, enrolled during the same time period. In closer detail, around 50% of the controls were outpatients, and the remaining were patients undergoing surgical interventions (laparoscopic cholecystectomy, appendicitis, and inguinal hernia) or admitted for a wide spectrum of other nonneoplastic conditions (elderly patients on physical-therapy rehabilitation after stroke or orthopedic injuries). Written informed consent was obtained from all study subjects. The study was conducted according to the Declaration of Helsinki and was approved by the Ethical Committee of Università Cattolica del Sacro Cuore.HCC cases and controls were interviewed by trained physicians using a structured questionnaire to collect information on demographics, medical history, and lifestyle habits including smoking history. Questions about lifestyle habits focused on the time period ending one year prior to diagnosis for cases and on the year prior to the interview date for controls.


### 2.2. Genotyping Methods

DNA was extracted from the peripheral blood lymphocytes of each participating subject.GSTM1 andGSTT1 null alleles were identified using a multiplex-polymerase chain reaction- (PCR-) based method [15]. The polymorphic site at nucleotide 638 in exon 7 (Arg213His (*2allele), rs9282861) of theSULT1A1 gene was genotyped by PCR-restriction fragment length polymorphisms (RFLP) analysis as described by Coughtrie et al. [16].CYP1A13′-flanking regionMspI polymorphism (CYP1A1*2A allele, rs4646903),CYP2E1 PstI/RsaI polymorphism (CYP2E1*5B allele, rs3813867 (PstI)), andCYP2E1 DraI (*5A or *6 alleles, rs6413432) were also determined by PCR-RFLP analyses. Quality control for each genotyping was performed in each experiment, and 10% of the total samples were randomly selected and reanalyzed with 100% concordance. All laboratory procedures were carried out blindly to case-control status.


### 2.3. Statistical Analysis

Hardy-Weinberg equilibrium (HWE) was tested for the control SNPs. Odds ratios (OR) of HCC and corresponding 95% confidence intervals (CI) according to analyzed polymorphisms were derived from unconditional multiple logistic regression models [17] using dominant model for carriers of the mutated allele, including terms for age and sex. When cell sizes were small (<5), exact logistic regression was used [18].We also examined the possible confounding effect of smoking, alcohol, and chronic infection with HBV and/or HCV. However, models including these covariates yielded very similar results; thus, given the small numbers in some strata, only the age- and sex-adjusted estimates were presented.Gene-gene interaction analysis was conducted, using as a reference group the homozygous wild-type individuals for both genes, while for gene-environment interaction analyses, the reference group was wild-type homozygotes not exposed to the environmental risk factor. Biological interaction between two genes was estimated using departure from additivity of effects as the criterion of interaction, as proposed by Rothman [19]. To quantify the amount of interaction, the attributable proportion (AP) due to interaction was calculated together with its 95% CI as described by Andersson et al. [20]. The AP due to interaction is the proportion of individuals among those exposed to the two interacting factors that is attributable to the interaction per se and it is equal to 0 in the absence of a biological interaction [19]. In order to test for more than multiplicative effect among two genes, the likelihood ratio test was used.The paper has been written according to the STREGA guidelines [21].


## 2.1. Study Population

Study participants were recruited among patients admitted to the teaching hospital “Agostino Gemelli” of the Università Cattolica del Sacro Cuore (Rome, Italy) from January 2005 until July 2010, and eligibility was restricted to white individuals born in Italy. Cases were 221 patients with HCC recruited among subjects referred to the Outpatient Liver Unit of the hospital. The diagnosis of HCC was performed according the AASLD guidelines [14]. The control group included 290 patients from the same hospital with a broad range of diagnoses, enrolled during the same time period. In closer detail, around 50% of the controls were outpatients, and the remaining were patients undergoing surgical interventions (laparoscopic cholecystectomy, appendicitis, and inguinal hernia) or admitted for a wide spectrum of other nonneoplastic conditions (elderly patients on physical-therapy rehabilitation after stroke or orthopedic injuries). Written informed consent was obtained from all study subjects. The study was conducted according to the Declaration of Helsinki and was approved by the Ethical Committee of Università Cattolica del Sacro Cuore.HCC cases and controls were interviewed by trained physicians using a structured questionnaire to collect information on demographics, medical history, and lifestyle habits including smoking history. Questions about lifestyle habits focused on the time period ending one year prior to diagnosis for cases and on the year prior to the interview date for controls.


## 2.2. Genotyping Methods

DNA was extracted from the peripheral blood lymphocytes of each participating subject.GSTM1 andGSTT1 null alleles were identified using a multiplex-polymerase chain reaction- (PCR-) based method [15]. The polymorphic site at nucleotide 638 in exon 7 (Arg213His (*2allele), rs9282861) of theSULT1A1 gene was genotyped by PCR-restriction fragment length polymorphisms (RFLP) analysis as described by Coughtrie et al. [16].CYP1A13′-flanking regionMspI polymorphism (CYP1A1*2A allele, rs4646903),CYP2E1 PstI/RsaI polymorphism (CYP2E1*5B allele, rs3813867 (PstI)), andCYP2E1 DraI (*5A or *6 alleles, rs6413432) were also determined by PCR-RFLP analyses. Quality control for each genotyping was performed in each experiment, and 10% of the total samples were randomly selected and reanalyzed with 100% concordance. All laboratory procedures were carried out blindly to case-control status.


## 2.3. Statistical Analysis

Hardy-Weinberg equilibrium (HWE) was tested for the control SNPs. Odds ratios (OR) of HCC and corresponding 95% confidence intervals (CI) according to analyzed polymorphisms were derived from unconditional multiple logistic regression models [17] using dominant model for carriers of the mutated allele, including terms for age and sex. When cell sizes were small (<5), exact logistic regression was used [18].We also examined the possible confounding effect of smoking, alcohol, and chronic infection with HBV and/or HCV. However, models including these covariates yielded very similar results; thus, given the small numbers in some strata, only the age- and sex-adjusted estimates were presented.Gene-gene interaction analysis was conducted, using as a reference group the homozygous wild-type individuals for both genes, while for gene-environment interaction analyses, the reference group was wild-type homozygotes not exposed to the environmental risk factor. Biological interaction between two genes was estimated using departure from additivity of effects as the criterion of interaction, as proposed by Rothman [19]. To quantify the amount of interaction, the attributable proportion (AP) due to interaction was calculated together with its 95% CI as described by Andersson et al. [20]. The AP due to interaction is the proportion of individuals among those exposed to the two interacting factors that is attributable to the interaction per se and it is equal to 0 in the absence of a biological interaction [19]. In order to test for more than multiplicative effect among two genes, the likelihood ratio test was used.The paper has been written according to the STREGA guidelines [21].


## 3. Results

The demographics, clinical features and lifestyle habits of 221 HCC cases and 290 controls are reported in Table1. Ever smokers were more common among cases, as well as infection with HBV and/or HCV (Table 1). The genotype frequencies were in HWE (P>0.05).Table 1
Distribution of 221 cases of hepatocellular carcinoma (HCC) and 290 controls according to selected factors.
HCC cases (N=221)
Controls (N=290)
N
(%)
N
(%)
Age (years)
<60
43
(19.5)
105
(36.2)
60–69
80
(36.2)
78
(26.9)
≥70
98
(44.3)
107
(36.9)
Sex
Male
160
(72.4)
176
(60.7)
Female
61
(27.6)
114
(39.3)
Smokinga
Never
85
(39.2)
173
(59.7)
Ever
133
(60.8)
117
(40.3)
Pb<0.001
Hepatitisa,c
No
64
(29.0)
282
(97.9)
Yes
157
(71.0)
6
(2.1)
Pb<0.001
aThe sum does not add up to the total because of some missing values.
b
P values from χ2 test.
cHepatitis was defined as history of hepatitis B and/or C.Table2 reports the distribution of the polymorphisms considered among HCC patients and controls. The carriers ofc2variant allele ofCYP2E1*5Bpolymorphisms were less common in cases (1.8%) than in controls (6.9%) corresponding to an OR of 0.23 (95% CI: 0.06–0.71). Similarly, the frequency of the variant allele ofCYP2E1*6polymorphism was also less common among cases (1.0%) than controls (10%), with an OR of 0.08 (95% CI: 0.01–0.33) (Table 2). The selected polymorphisms ofCYP1A1,GSTM1,GSTT1, andSULT1A1 did not significantly influence susceptibility to HCC.Table 2
Distribution of cases and controls, odds ratiosa (OR), and 95% confidence intervals (CI) for hepatocellular carcinoma (HCC) according to selected polymorphisms.
HCC cases (N=221)
Controls (N=290)
OR (95% CI)
N
(%)
N
(%)
CYP1A1
*
2A
wt/wt
165
(74.7)
226
(77.9)
1b
wt/mt and mt/mt
56
(25.3)
64
(22.7)
1.21 (0.80–1.84)
CYP2E1
*
5B
c1/c1
217
(98.2)
270
(93.1)
1b
c1/c2 and c2/c2
4
(1.8)
20
(6.9)
0.23 (0.06–0.71)
CYP2E1
*
6
c
wt/wt
204
(99.0)
261
(90.0)
1d
wt/mt and mt/mt
2
(1.0)
29
(10.0)
0.08 (0.01–0.33)
GSTM1
c
Present
96
(47.8)
139
(48.1)
1b
Null
105
(52.2)
150
(51.9)
0.99 (0.68–1.43)
GSTT1
c
Present
141
(70.1)
220
(76.1)
1b
Null
60
(29.9)
69
(23.9)
1.35 (0.89–2.05)
SULT1A1
wt/wt
132
(59.7)
180
(62.1)
1b
wt/mt and mt/mt
89
(40.3)
110
(37.9)
1.22 (0.84–1.77)
aAdjusted for age and sex.
bReference category.
cThe sum does not add up to the total because of some missing values.
dCalculated from exact logistic regression analysis.
wt: wild-type allele.
mt: variant-type allele.The gene-gene and gene-smoking interaction results are reported in Tables3 and 4. We observed a borderline increased risk for HCC among carriers of combinedCYP1A1*2A andSULT1A1variant alleles as compared to the double wild-type homozygotes (OR = 1.67; 95% CI: 0.97–3.24) (Table 3). A significant interaction was reported betweenGSTT1 and smoking (AP = 0.48; 95% CI: 0.001–0.815), with an OR of 3.13 (95% CI: 1.69–5.82) forGSTT1 null genotype carriers who were smokers (Table 4). A borderline significant interaction was also observed forSULT1A1 and smoking (AP = 0.36; 95% CI: −0.021–0.747), with an OR of 3.05 (95 CI: 1.73–5.40) for thoseSULT1A1 variant allele carriers who were smokers (Table 4).Table 3
Effect of the genes-gene interaction on the development of hepatocellular carcinoma.
Cases : controls
ORa (95% CI)
P
b
AP (95% CI)
GSTM1 × CYP1A1*2A
Present/wt homozygote
74 : 113
1c
Null/wt homozygote
78 : 112
1.05 (0.69–1.61)
Present/any mt
22 : 26
1.32 (0.69–2.53)
Null/any mt
27 : 38
1.04 (0.58–1.88)
0.521
nc
GSTM1 × GSTT1
Present/present
66 : 103
1c
Null/present
75 : 117
0.97 (0.63–1.50)
Present/null
30 : 36
1.28 (0.71–2.30)
Null/null
30 : 33
1.39 (0.77–2.53)
0.773
0.109 (−0.603; 0.820)
GSTM1 × SULT1A1
Present/present
53 : 84
1c
Null/present
67 : 96
1.09 (0.68–1.76)
Present/null
43 : 55
1.40 (0.81–2.41)
Null/null
38 : 54
1.22 (0.70–2.13)
0.566
nc
GSTT1 × CYP1A1*2A
Present/wt homozygote
108 : 169
1c
Null/wt homozygote
44 : 56
1.20 (0.74–1.93)
Present/any mt
33 : 51
0.98 (0.58–1.63)
Null/any mt
16 : 13
2.00 (0.91–4.41)
0.289
0.414 (−0.149; 0.976)
GSTT1 × SULT1A1
Present/wt/wt
81 : 138
1c
Null/wt/wt
39 : 42
1.59 (0.94–2.70)
Present/any mt
60 : 82
1.40 (0.90–2.20)
Null/any mt
21 : 27
1.48 (0.77–2.84)
0.347
nc
SULT1A1 × CYP1A1*2A
wt homozygote/wt homozygote
99 : 138
1c
mt carrier/wt homozygote
66 : 88
1.14 (0.75–1.74)
wt homozygote/mt carrier
33 : 62
1.09 (0.64–1.85)
mt carrier/mt carrier
23 : 22
1.67 (0.97–3.24)
0.493
0.269 (−0.330; 0.867)
OR: odds ratio; CI: confidence interval; AP: attributable proportion; nc: not calculable.
aAdjusted for age and sex.
b
P from test for multiplicative interaction.
cReference category.
wt: wild-type allele.
mt: variant-type allele.Table 4
Effect of the gene-smoking interaction on the development of hepatocellular carcinoma.
Cases : controls
ORa (95% CI)
P
b
AP (95% CI)
CYP1A1
*
2A × smokingc
wt homozygote/no
65 : 136
1d
wt homozygote/yes
98 : 90
2.16 (1.29–3.36)
mt carrier/no
20 : 37
1.08 (0.58–2.03)
mt carrier/yes
35 : 27
2.81 (1.51–5.23)
0.680
0.201 (−0.328; 0.730)
GSTM1 × smokingc
Present/no
31 : 91
1d
Present/yes
62 : 48
4.01 (2.20–7.28)
Null/no
48 : 81
1.82 (1.05–3.18)
Null/yes
57 : 69
2.34 (1.32–4.23)
0.004
nc
GSTT1 × smokingc
Present/no
59 : 129
1d
Present/yes
81 : 91
1.86 (1.17–2.97)
Null/no
20 : 43
0.99 (0.53–1.86)
Null/yes
38 : 26
3.13 (1.69–5.82)
0.230
0.480 (0.001; 0.815)
SULT1A1 × smokingc
wt homozygote/no
52 : 103
1d
wt homozygote/yes
78 : 77
1.93 (1.19–3.14)
mt carrier/no
33 : 70
1.01 (0.59–1.75)
mt carrier/yes
55 : 40
3.05 (1.73–5.40)
0.250
0.363 (−0.021; 0.747)
OR: odds ratio; CI: confidence interval; AP: attributable proportion; nc: not calculable.
aAdjusted for age and sex.
b
P from test for multiplicative interaction.
cThe sum does not add up to the total because of some missing values.
dReference category.
wt: wild-type allele.
mt: variant-type allele.


## 4. Discussion

Our study identifiedCYP2E1*5B andCYP2E1*6variant alleles associated with a reduced risk of HCC. There was a borderline increased risk for HCC among carriers of combinedSULT1A1 andCYP1A1*2A variant alleles. The polymorphisms inGSTT1 andSULT1A1are associated with increased susceptibility to smoking-related HCC.CYP2E1 can activate N-nitrosamines and benzene contained in cigarette smoke [22] and is involved in alcohol-mediated generation of oxidative stress [23]. The expression ofCYP2E1 correlates with the generation of hydroxyethyl radicals and lipid peroxidation products [23]. The variantCYP2E1*5B andCYP2E1*6 alleles are associated with increased transcription ofCYP2E1[24] that leads to development of HCC by promoting carcinogenesis.No significant association betweenCYP2E1*6 and HCC was reported so far [25, 26], while contradictory results were reported for theCYP2E1*5B variant allele [27–35]. A recent meta-analysis did not findCYP2E1*5B c2 allele to be associated with HCC [36], also after stratifying among Asians and white. Studies conducted among white individuals, however, were few [26, 30, 35] and included limited numbers of cases.The favorable effect of bothCYP2E1 polymorphisms on HCC development is not consistent with the biological premises implying a promoting role of a high activity enzyme. However, the only study previously conducted in an Italian population [35] onCYP2E1*5B c2 allele and HCC did report a similar association, indicating a favorable role of the variant allele against HCC which deserves further investigation.Our results suggest that up to 48% and 36% of HCC cases among smokers carrying, respectively, variantGSTT1andSULT1A1 alleles occurred because of gene-smoking interaction. However, we could not further stratify these two results according to quantity of smoking, as numbers were low. Smoking is recognized as a risk factor for HCC [3–5] and, together with HBV and HCV, one of the major risk factors in Europe [37], and enzymes coded byGSTT1 andSULT1A1 have their role in the metabolism of tobacco carcinogens. There is therefore a biological ground for possible synergic carcinogenic effect.There was a borderline synergic effect ofSULT1A1 andCYP1A1*2A polymorphisms in HCC carcinogenesis. The effect ofSULT1A1 polymorphism on HCC development has not been investigated so far. It has been reported, however, that an enzyme coded bySULT1A1 variant allele has twofold lower catalytic activity in detoxifying the procarcinogens [38]. The biological significance of variantCYP1A1*2Avariant allele is uncertain, butCYP1A1*2Ahas been reported to increase susceptibility to several cancer types, including lung, breast, and cervical cancer [39–41]. There is therefore a rationale for a synergic effect of these two SNPs in HCC carcinogenesis.In our study, there was no significant association between HCC and alcohol. As most of the cases have hepatitis, this could lead them to stop drinking. Consequently we were unable to perform gene-alcohol interaction analysis. Secondly, we cannot exclude a selection bias. However, since the observed frequencies of the variant alleles ofCYP1A1*2A, as well as the null genotypes ofGSTM1 andGSTT1, were in line with those previously reported in the Italian population, a major impact of such bias is unlikely [35, 42]. Thirdly, our study was underpowered to perform gene-gene and gene-interaction analysis.In conclusion, we report thatCYP2E1*5B andCYP2E1*6polymorphisms have a favorable effect on the development of HCC, while the polymorphisms inGSTT1andSULT1A1may increase HCC susceptibility among smokers.



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