Association between xenobiotic gene polymorphisms and non-Hodgkin's lymphoma risk
Dr Andrew Spencer, BMT Programme, Alfred Hospital, Commercial Road, Prahran, Victoria, Australia 3181. E-mail: email@example.com
Summary. The last four decades have seen a significant increase in the incidence of non-Hodgkin's lymphoma (NHL) as a possible result of increasing environmental carcinogen exposure, particularly pesticides and solvents. Based on the increasing evidence for an association between carcinogen exposure-related cancer risk and xenobiotic gene polymorphisms, we have undertaken a case–control study of xenobiotic gene polymorphisms in individuals with a diagnosis of NHL. Polymorphisms of six xenobiotic genes (CYP1A1, GSTT1, GSTM1, PON1, NAT1, NAT2) were characterized in 169 individuals with NHL and 205 normal controls using polymerase chain reaction-based methods. Polymorphic frequencies were compared using Fisher's exact tests, and odds ratios for NHL risk were calculated. Among the NHL group, the incidence of GSTT1 null and PON1 BB genotypes were significantly increased compared with controls, 34%vs 14%, and 24%vs 11% respectively. Adjusted odds ratios calculated from multivariate analyses demonstrated that GSTT1 null conferred a fourfold increase in NHL risk (OR = 4·27; 95% CI, 2·40–7·61, P < 0·001) and PON1 BB a 2·9-fold increase (OR = 2·92; 95% CI, 1·49–5·72, P = 0·002). Furthermore, GSTT1 null combined with PON1 BB or GSTM1 null conferred an additional risk of NHL. This is the first time that a PON1 gene polymorphism has been shown to be associated with cancer risk. We conclude that the two polymorphisms, GSTT1 null and PON1 BB, are common genetic traits that pose low individual risk but may be important determinants of overall population NHL risk, particularly among groups exposed to NHL-related carcinogens.
The incidence of non-Hodgkin's lymphoma (NHL) has been steadily rising since the 1950s (Rabkin et al, 1993; Nordstrom, 1996; Cartwright et al, 1999) and it has been suggested that greater environmental carcinogen exposure, particularly exposure to pesticides and solvents, may be responsible (Cantor, 1982; Olsson & Brandt, 1988; Wigle et al, 1990; Hardell & Eriksson, 1999). Implicated pesticides include phenoxyacetic acid herbicides, organophosphate insecticides, triazine herbicides and fungicides. Furthermore, the risks of developing NHL associated with agricultural pesticide exposures have been found to be greater among women with a family history of cancer, particularly lymphatic or haemopoietic malignancy among first-degree relatives (Zahm et al, 1993). Such a familial cancer risk may reflect shared environmental exposure or it may be a crude indicator of heritable genetic susceptibility to the carcinogenic effects of chemical exposures.
All higher species have evolved complex xenobiotic enzyme systems for protection against environmental genotoxins (Nebert et al, 1996). The human system comprises two main classes of enzymes: Phase 1 cytochrome P-450 (CYP) enzymes that metabolically activate procarcinogens to genotoxic electrophilic intermediates, and Phase II enzymes, including glutathione S-transferases (GSTT1 and GSTM1), paraoxonase (PON1) and N-acetyltransferases (NAT1 and NAT2) which principally conjugate the intermediates to excretable hydrophilic derivatives, completing the detoxification cycle. Polymorphisms of several of these genes are believed to be key factors in determining cancer susceptibility to toxic or environmental chemicals (Nebert et al, 1996; Perera, 1997).
Paraoxonase (PON1), an enzyme expressed principally in the liver, plays an important role in the detoxification of organophosphates processed through the P450/PON1 pathway and exhibits a substrate-dependent polymorphism with two known alleles producing an A (Gln192 isoform) or B (Arg192 isoform) genotype (Costa et al, 1999; Hegele, 1999). Animal studies have demonstrated that PON1 activity levels play a major role in determining sensitivity to organophosphates and it has been postulated, but never demonstrated, that an individual's PON1 phenotype may determine their susceptibility to insecticide-linked tumours (Costa et al, 1999).
To ascertain whether there is any association between xenobiotic gene polymorphisms and NHL risk, we determined the frequencies of GSTT1, GSTM1, NAT1, NAT2, PON1 and CYP1A1 gene polymorphisms in the general Australian population and compared them with the frequencies found in a group of NHL patients.
Patients and methods
Subjects. Lymph node or bone marrow biopsy specimens were obtained from 169 Caucasian individuals with confirmed diagnoses of follicle centre cell (FCC) (n = 60), diffuse large cell (DLC (n = 60) or other subtypes (n = 49) of non-Hodgkin's lymphoma (NHL). The control group comprised 205 sex-matched healthy Caucasian volunteer bone marrow donors. The research protocol was approved by the Hunter Area Health Service Research Ethics Committee and informed consent was obtained from all living participants.
Genotyping. Genomic DNA was purified using salt extraction followed by ethanol precipitation (Miller et al, 1988). All genotyping was performed by polymerase chain reaction (PCR) or PCR-restriction length fragment polymorphism (RLFP) using a microtube thermocycler (Corbett Research, Sydney, Australia). Presence of the CYP1A1 gene mutation, m1, involving either or both of the CYP1A1 alleles, was identified using a previously described PCR method with subsequent Msp1 restriction enzyme digestion (Cascorbi et al, 1996). A previously described method wasalso used to amplify and analyse GSTM1 and GSTT1, with the absence of amplifiable GSTM1 or GSTT1 indicatingthe null genotype for each gene respectively (Chen et al, 1996). For PON1, a multiplex PCR containing two forward primers specific for either the Gln192 isoform (PON1 A) or the Arg192 isoform (PON1 B) was performed as published (Schmitz & Lindpaintner, 1993; Serrato & Marian, 1995). NAT1 allellic variants were distinguished by detectable changes in the 3′-region of the NAT1 gene following PCR and MboI1 digestion as described (Bell et al, 1995). Finally, an established PCR-RFLP method was used to determine NAT2 genotype (Abe et al, 1993). In all PCR assays, appropriate negative and positive controls were included.
Statistical analysis. The frequency of the various enzyme genotypes in NHL cases and controls were compared using Fisher's exact tests (two-tailed). Odds ratios (OR) express the relative risk of NHL with a specific genotype and are calculated by dividing the odds of an NHL patient having a specific genotype by the odds of a control subject having the same genotype. The crude ORs were calculated, along with 95% confidence intervals. In addition, Fisher's exact tests were used to examine for significant differences in the frequency of genotypes between DLC, FCC and ‘other’ NHL cases. An unconditional multivariate logistical analysis producing adjusted ORs was performed to determine which of the genotypes continued to show significant differences between NHL cases and controls in the presence of others. All genotypes studied were included as covariables.
The observed genotype frequencies in our control population was in accordance with data from other published studies (Bell et al, 1995; Serrato & Marian, 1995; Chen et al, 1997; Krajinovic et al, 1999; Agundez et al, 2000). This suggests that any difference between the genotype frequencies within the NHL group and the control group were not related to a sampling problem with the latter. The frequency of GSTT1, GSTM1, NAT1, NAT2, PON1 and CYP1A1 genotypes in NHL cases and controls are presented in Table I. The incidence of GSTT1 null and PON1 BB genotypes were significantly increased in NHL cases compared with controls. Among NHL cases, 34% were GSTT1 null, compared with 14% of controls; PON1 BB was present in 24% of NHL cases, compared with 11% of controls (Table I). Consequently, the null genotype for GSTT1 conferred a threefold increase in the risk of NHL (OR = 3·22; 95% CI, 1·94–5·34) and PON1 BB a 2·5-fold increase in the risk of NHL (OR = 2·50, 95% CI, 1·37–4·55). No significant differences were found in the frequency of GSTM1, NAT1, NAT2 and CYP1A1 genotypes in NHL cases and controls (Table I). In addition, no significant differences were found in the distribution of genotypes in DLC, FCC and ‘other’ NHL cases. Multivariate analysis verified the findings of the univariate analyses. GSTT1 and PON1 continued to show significant differences between NHL cases and controls in the presence of other variables. Multivariate adjusted ORs for GSTT1 null and PON1 BB were 4·27 (95% CI, 2·40–7·61) and 2·92 (95% CI, 1·49–5·72) respectively (Table II). No other significant differences were found.
Table I. Comparison of GSTT1, GSTM1, NAT1, NAT2, PON1 and CYP1A1 genotype frequencies in cases and controls (univariate analysis).
|GSTT1||Present||112||66||177||86||1.00|| || |
|GSTM1||Present||78||46||95||46||1·00|| || |
|NAT1‡||Rapid||76||46||83||43||1.00|| || |
|NAT2§||Rapid||71||43||97||51||1·00|| || |
|PON1||AA||73||45||103||52||1.00|| || |
|PON1||AA/AB||123||76||177||89||1·00|| || |
|CYP1A1||–/–||143||85||159||82||1.00|| || |
Table II. Adjusted odds ratios (multivariate analysis) *.
|GSTT1||Present||1·00|| || |
|GSTM1||Present||1·00|| || |
|NAT1||Rapid||1·00|| || |
|NAT2||Rapid||1·00|| || |
|PON1||AA||1·00|| || |
|CYP1A1||–/–||1·00|| || |
|–/+, +/+||0·87||0·47–1·61|| 0·654|
We also investigated whether the prevalence of combined GSTT1 null and GSTM1 null genotype (double-null genotype) was significantly increased in NHL cases compared with controls, as this has previously been reported in patients with acute lymphoblastic leukaemia (ALL) (Chen et al, 1997). Among NHL cases, 18·5% were both GSTT1 and GSTM1 null, compared with 6·4% of controls (Table III). When individuals with both GSTT1 and GSTM1 were considered as the reference group, a multivariate analysis demonstrated an increased risk (OR = 3·6; 95% CI, 1·74–7·44) in individuals with the double-null genotype. The risk was higher than that seen with GSTTI null alone (OR = 2·5; 95% CI, 1·22–5·17). Similarly, we investigated whether the risk of NHL was increased further when the identified risk genotypes of GSTT1 null and PON1 BB were combined. As no significant difference was previously found in the associated risk of AA and AB PON1 genotypes, the genotypes of AA and AB were combined for this analysis. When individuals without either of the risk-elevating genotypes were considered as the reference group (i.e. those with GSTT1 present and with PON1 genotypes of AA or AB) multivariate analyses demonstrated a substantially increased risk (OR = 9·1; 95% CI, 2·71–30·36) in individuals carrying both risk genotypes (Table IV).
Table III. Combined effects of GSTT1 and GSTM1 null genotypes *.
|Present||Present||53||31·5||80||39·2||1·00|| || |
|Null||Present||25||14·9||15|| 7·4||2·52||1·22–5·17|| 0·018|
|Null||Null||31||18·5||13|| 6·4||3·60||1·74–7·44||< 0·001|
Table IV. Combined effect of GSTT1 null and PON1 BB genotypes *.
|None||Present||A/A or A/B||80||49||156||78||1·00|| || |
|One||Null||A/A or A/B||43||27|| 21||11||3·99||2·33–7·15||< 0·001|
|Two||Null||B/B||14||9|| 3||2||9·10||2·71–30·36||< 0·001|
It is generally believed that genetic factors and environmental exposures predispose individuals to lymphomagenesis. Therefore, in an effort to increase our understanding of the interaction between potential environmental exposures and genetic factors in NHL we determined the frequencies of CYP1A1, GSTM1, GSTT1, NAT1, NAT2 and PON1 gene polymorphisms in a control population and a population of patients with NHL.
The GSTT1 null and PON1 BB genotypes were significantly increased in NHL cases compared with controls, with multivariate analyses showing GSTT1 null conferring a 4·3-fold increase in the risk of NHL and PON1 BB conferring a 2·9-fold increase. The double-null genotype for GSTM1 and GSTT1, and the combination of GSTT1 null and PON1 BB conferred an even greater risk with adjusted ORs of 3·6 and 9·1 respectively. Although the GSTM1 null genotype by itself was not associated with an increased risk of NHL, it demonstrated an interaction with the GSTT1 null genotype to confer a higher risk than the GSTT1 null genotype alone. This supports the notion that a complex interaction of factors, both endogenous and exogenous, is probably involved in predisposition for the development of NHL. Furthermore, the double-null genotype for GSTM1 and GSTT1 has been shown to be significantly more frequent among black children with ALL (Chen et al, 1997). Our findings are consistent with the latter, now demonstrating a second group of lymphoid malignancies associated with the double-null GSTM1 and GSTT1 phenotype. This raises the possibility of common aetiological mechanisms underlying some cases of both NHL and ALL, possibly shared environmental exposures interacting with the at-risk double-null genotype.
We have demonstrated for the first time ever an association between PON1 polymorphisms and cancer in humans. Intuitively this is not surprising in the context of NHL risk. PON1 plays a major role in the detoxification of organophosphate compounds processed through the P450/PON1 pathway, and the development of NHL has been associated in epidemiological studies with pesticide exposure (Cantor, 1982; Wigle et al, 1990; Hardell & Eriksson, 1999). It is therefore possible that the PON1 BB genotype may predispose to delayed hydrolysis of a genotoxic metabolic intermediary of organophosphate detoxification thus contributing to lymphomagenesis. Alternatively, it is now becoming apparent that other classes of substrates for PON1 exist (Billecke et al, 2000), including large numbers of endogenous compounds and it may be that PON1 plays an essential role in the detoxification of endogenously generated genotoxins. It is also uncertain whether PON1 may have any role in the detoxification of the N-methylcarbamate ester non-organophosphate insecticides that have been shown to have in vitro mutagenic effects (Wang et al, 1998).
The two polymorphisms that we have identified, GSTT1 null and PON1 BB, are common genetic traits that pose low individual risk but may be important determinants of overall population NHL risk, particularly among groups exposed to NHL-related carcinogens. The previously unreported identification of the PON1 BB gene polymorphism as a risk factor for NHL is of particular interest in view of the role that the paraoxonase gene family plays in the detoxification of organophosphates.
This work was funded in part by the McCaughey Research Scholarship (I.K.) and The Anderson Trust (A.S.). We thank Rhonda Holdsworth and Steven Braye for assistance in providing control and patient specimens.