Effects of tobacco on the DNA of smokers and non-smokers affected by OSCC: systematic review and meta-analysis

SCHUCH, Lauren Frenzel; VIANA, Karolina Skarlet Silva; DE ARRUDA, José Alcides Almeida; ABREU, Lucas Guimarães; AGUIAR, Maria Cássia Ferreira de; BERNARDES, Vanessa Fátima

doi:10.1590/1807-3107bor-2023.vol37.0008

Lauren Frenzel SCHUCH

Universidade Federal de Minas Gerais – UFMG, School of Dentistry, Department of Oral Surgery and Pathology, Belo Horizonte, MG, Brazil.

http://orcid.org/0000-0002-0993-936X

Karolina Skarlet Silva VIANA

Universidade Federal de Minas Gerais – UFMG, School of Dentistry, Department of Oral Surgery and Pathology, Belo Horizonte, MG, Brazil.

http://orcid.org/0000-0001-6467-1434

José Alcides Almeida DE ARRUDA

Universidade Federal de Minas Gerais – UFMG, School of Dentistry, Department of Oral Surgery and Pathology, Belo Horizonte, MG, Brazil.

http://orcid.org/0000-0002-6599-3950

Lucas Guimarães ABREU

Universidade Federal de Minas Gerais – UFMG, School of Dentistry, Department of Child’s and Adolescent’s Oral Health, Belo Horizonte, MG, Brazil.

http://orcid.org/0000-0003-2258-8071

Maria Cássia Ferreira de AGUIAR

Universidade Federal de Minas Gerais – UFMG, School of Dentistry, Department of Oral Surgery and Pathology, Belo Horizonte, MG, Brazil.

http://orcid.org/0000-0001-5134-3466

Vanessa Fátima BERNARDES

Universidade Federal de Minas Gerais – UFMG, Biological Sciences Institute, Department of Pathology, Belo Horizonte, MG, Brazil.

http://orcid.org/0000-0003-0194-7434

Corresponding Author: Vanessa Fátima Bernardes E-mail: bernardesvf@icb.ufmg.br

Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.

Author	Sample	Smoker		Gene/Protein	Method	Results
Author	Sample	Yes	No	Gene/Protein	Method	Results
Katoh et al.³³	62	39	23	NAT1 e 2	PCR	Individuals with NAT1*10 alleles are at higher risk for oral squamous cell carcinoma, but smoking history may not play a role in this genetic relationship. The role of NAT1 appears to be independent of smoking behavior.
Tanimoto et al.³⁴	100	58	42	CYP1A1, GSTM1	PCR	The ORs of the individual genotypes increases as cigarette dose increases; however, the genetic difference in susceptibility was largest with ORs of 7.0 for genotype m2/m2 at the lower smoking level. These results indicate that the patients with m2/m2 contracted oral squamous cell carcinoma from a significantly lower cigarette dose than those with other genotypes. The fact that there is less difference in susceptibility among the genotypes at high cigarette dose levels may be ascribable to a saturation of the metabolic response. On the other hand, the amount of life-time alcohol consumption did not show a significant difference in the distribution of genotypes of either CYP1A1 or GSTM1, when estimated by drinking index (DI=the amount of alcohol converted into ethanol per day x the number of years of drinking).
Park et al.⁴⁰	164	147	17	GSTM1 e 3	PCR	For African-American subjects who smoked more than 24 PY, risk for oral cancer was significantly associated with the GSTM1 null polymorphism (OR: 5.4, 95%CI: 1.2 ± 24). No association was observed in African-Americans who were light-smokers (i.e. 24 PY). A test for interaction between smoking and the GSTM1 genotype was not significant when the smoking-GSTM1 genotype interaction variable was introduced into the multiple logistic regression model for oral cancer risk. Significant associations were not observed between the GSTM3 genotype and oral cancer risk in African-Americans after stratification by smoking dose, although a trend was observed between the GSTM3 (B/B) genotype and oral cancer risk in the light-smoking African-American group (OR: 0.19, 95%CI: 0.03 ± 1.3).
Hsieh et al.²⁰	187	182	5	p53	PCR	A specific pattern of mutation was observed in exons 5–9 of the p53 gene in OSCCs from smokers, alcohol users and BQ chewers. G:C to A:T transitions were the predominant mutations observed and associated with BQ and tobacco use. Seventeen of the 18 (94.44%) frameshift mutations including deletions and insertions occurred in smokers. Among them, 14 patients (82.35%) were also BQ chewers. In addition, most (20/22, 90.91%) G:C to T:A transversions occurred in smokers. All A:T to T:A and G:C mutations (n = 11) occurred in BQ chewers. All G:C to C:G transversions occurred in either smokers or BQ chewers. All of the mutations identified in patients with OSCCs in this series were somatic and not germ-line in origin, as DNA from normal tissue adjacent to p53-mutated tumor was negative for p53 mutations by both PCR–SSCP and DNA sequencing analysis.
Kietthubthew et al.³⁵	53	50	3	GSTM1, GSTT1	PCR	The GSTM1 null genotype had a significant effect on oral cancer risk (OR: 3.0, 95%CI: 1.4–6.7), whereas the GSTT1 revealed no association (OR: 0.6, 95%CI: 0.3–1.3). The effect of the GST-susceptible genotypes on oral cancer risk was not increased with the combined deletion of GSTM1 and GSTT1 (OR: 2.0, 95%CI: 0.5–7.8). The GSTM1 wild type and GSTM1 null genotypes had no influence on oral cancer among nonsmokers and occasional smokers. However, frequent smokers with the GSTM1 null had a significantly increased risk for oral cancer (OR: 4.1; 95%CI: 1.5–11.3). With respect to the betel-chewing habit, the GSTM1 null increased the risk among frequent chewers (OR: 4.0; 95%CI: 1.3–12.9). Interestingly, for individuals who chew betel without smokeless tobacco, the risk was raised to 22-fold (95%CI: 2.2–222.0).
Chaves et al.³⁷	71	66	5	p53	PCR	The presence of TP53 mutation was independent of tobacco consumption. There was a predominance of A-G (45%) substitutions in current smokers and drinkers, followed by A-C transversions in 20% of the cases. The G-T mutation characteristic of tobacco smoke was found in only 1 tumor. Non-smokers and non-alcohol users were found to have 42.8% of G-A mutations, 2 of them were located in CpG sites and 1 in non-CpG sites.
Kietthubthew et al.³⁶	106	72	34	XRCC1 e 3, XPC, XPD	PCR-RFLP	The homozygous variant genotype of XRCC1 399Gln reduced the OSCC risk (OR: 30, 95%CI: 0.1-0.88, p=0.03). The heterozygous genotypes for XRCC1 194Trp and XRCC3 241Met significantly increased the risk (OR: 2.26, 95%CI: 1:20-4.28, p = 0.01; OR: 2.31, 95%CI: 1.09-4.91, p = 0.03, respectively). There was a marginally higher risk for the heterozygous XPD exon 6 (OR: 1.74, 95% CI =0.94-3.22, p =0.08).
Korabiowska et al.³⁹	40	28	12	Ku70, Ku80	IHC	In carcinomas from smokers, Ku70-positive cells were found in 82.9% of tumors and Ku80 positivity was observed in 85.7% of carcinomas. The maximum values of Ku70 and Ku80 expressions in carcinomas from smokers reached 60% and 50%, respectively. In tumors from non-smokers, Ku70 positivity was observed in 87.5% of cases and Ku80 positivity in 93.8% of tumors. Ku70 and Ku80 expression values reached maxima of 40%. The comparison of Ku70 and Ku80 expressions in tumors from smokers and non-smokers demonstrated a highly significant result for Ku70 (p = 0.008). Significant correlations between Ku70 and Ku80 expression were found in carcinomas from non-smokers (p < 0.05). In tumors from smokers, these significant relationships were not preserved (p > 0.05).
Prior et al.³⁸	27	21	6	ND2	PCR	For ND2 gene, nucleotide 4917 was a significant mutation hotspot (P 1/4 0.027) and thus a potential smoking-associated biomarker in oral SCC. All patients having a mutation were males and classed as smokers with the exception of patient 5 whose smoking status was not known. Seven different types of mutation were discovered in the region of the D-Loop between nt 8 and 429. Base substitutions were observed in 16 (53.3%) different patients, 15 of whom had a classified smoking status. Of these, the 10 male patients with mutations were all self-classified smokers whereas, conversely, 4 of the 5 females with mutations were self-classified as non-smokers. This association of sex (males) and smoking status was statistically significant (p = 0.003) for patients with mutations.
Sharma et al.²⁷	40	18	22	GSTM1, GSTT1	PCR	The prevalence of the GSTM1 null genotype in cancer cases was 52.5% (21/40). A total of 42.5% (17/40) of oral cancers had homozygous deletion of GSTT1 genotype as compared to 14.9% (13/87) of the controls. Only 14 individuals with cancer were heavy smokers (> 40 pack years) and 7 were alcohol consumers. Four individuals were occasional smokers and the rest were non-smokers. Three individuals were pan/tobacco chewers. The GSTM1 null genotype prevalence was 35.71% (5/14) in smokers. In the case of GSTT1, the differences between oral cancer cases and control smokers were significant (p = 0.04) (OR: 6.33; 95%CI: 1.0-44.1).
Anantharaman et al.²⁸	458	391	67	CYP1A1, GSTM1, GSTT1	PCR	Among mixed habits of tobacco (chewers and smokers), CYP1A1 MspI homozygous variant genotype (m2/m2) contributed a 3.2-fold increased risk to OSCC [95% CI, 1.10–10.28; p=0.05]. GSTM1 null genotype shows a 1.29 times greater risk for OSCC (95% CI, 1.04– 1.65; P=0.05). GSTT1 null genotype offered protection to OSCC. Individuals carrying this genotype were at 0.5 times reduced risk for cancer conditions (95%C: 0.39–0.83; p =0.004). Among tobacco chewers, GSTT1 null genotype offered protection by decreasing the risk for OSCC by 0.27-fold (95%CI: 0.14–0.53; p = 0.0001).
Bau et al.¹³	154	137	17	XPA A-23G, XPD Lys751Gin	PCR	The crude OR of the stratification with either harboring variant XPA genotype (A/G or G/G, “variant”) or with smoking habit was 3.52 (95%CI: 1.26-9.84), and the crude OR of those with both harboring variant XPA genotype and smoking habit was increased to 47.7 (95%CI: 15.48-147.01). By the same analyses strategy, the same trend was observed and the joint effect of XPD genotype and smoking habit on oral cancer were also significant. The “common” group with putative low-risk XPD A/A genotype and without smoking habit was used as reference. The crude OR of the stratification with either harboring variant XPD genotype (A/C or C/C, “variant”) or with smoking habit was 28.48 (95%CI: 13.93-58.23), and the crude OR of those with both harboring variant XPD genotype and smoking habit was 26.33 (95%CI: 7.87-88.04). The crude ORs of the stratification with one of the three factors, variant XPA (A/G or G/ G), variant XPD (A/C or C/C) genotype, or smoking habit, was 3.59 (95%CI: 1.27-10.19), and the crude ORs of the stratification with two or all of the three factors were significantly increased to 24.05 (95%CI: 8.38-68.95). The 7-fold synergistic increase from 3.59 to 24.05 suggested that genetic factors (XPA and XPD), modified by the environmental factor (smoking), may also contribute to oral cancer risk.
Chen et al.²¹	78	61	17	hTERT	IHC	OSCC patients with areca quid chewing (p=0.029), cigarette smoking (p=0.027), or alcohol drinking habits (p = 0.025) were prone to have a higher mean cytoplasmic hTERT labeling score in OSCC samples than OSCC patients without these oral habits. OSCC patients with all 3 oral habits (p = 0.005) or with at least one oral habit (p = 0.007) also had a significantly higher cytoplasmic hTERT LS than OSCC patients without any oral habit.
Tsai et al.²²	680	512	168	Exo1	PCR	The Exo1 K589E was associated with higher susceptibility of oral cancer. The allele frequency distributions of the Exo1 K589E showed that A allele of Exo1 K589E was associated with higher susceptibility for oral cancer, while others were not. Genotype distribution of various genetic polymorphisms of exo1 K589E was significantly different between oral cancer and control groups with smoking habit (p = 2.41E-11). Distributions of Exo1 K589E A homozygote/heterozygote and G homozygote in controls and oral cancer patients with no smoking habit were 76/116 and 59/109, respectively (p = 0.344, OR: 0.795, 95%CI: 0.519-1.218).
Anantharaman et al.²⁹	665	325	340	GSTM1null, GSTT1null	PCR	Increased risk of oral cancer was associated with rs4646903 (OR: 1.66; 95%CI: 1.16-2.43), GSTM1 null (OR: 1.50; 95%CI: 1.20-1.87) and GSTT1 null genotype (OR: 0.47; 95%CI: 0.32-0.69). Additionally, rs2031920 and rs3813867 (OR: 0.39; 95%CI: 0.18-0.86) and rs1381 (OR: 0.75; 95%CI: 0.63-0.89) significantly reduced the risk of oral cancer. rs2031920 and rs3813867 significantly reduced oral cancer risk among exclusive tobacco chewers, (OR = 0.13; 95%CI = 0.03–0.59) and a positive dose-response relationship was observed. Similar results were observed for rs13181 (OR = 0.76; 95% CI = 0.59–0.97).
Tsai et al.²³	213	159	54	CCND1	PCR	The genotype distribution of the polymorphisms of CNND1 A870G was significantly different between oral cancer patients and controls with a smoking habit (p = 0.0006). The GG genotype frequency was still significantly lower (12.9%) in cancer patients with a smoking habit than in smoking controls (16.6%).
Mallick et al.³⁰	39	31	8	p53, BCL-XL	IHC	In patients with tobacco habits (chewers + smokers), increased p53 intensity (p = 0.063) was observed compared to those with no habits, although it did not reach statistical significance. The probability of treatment failure (hazard ratio) was 3.2 times higher in the unfavorable responders compared to that of favorable response group. Bcl-xL protein was significantly upregulated (p=0.048) in the unfavorable responders compared to the favorable responders.
Tsai et al.²⁴	230	168	62	hOGG1	PCR	The genotype distribution of hOGG1 codon 326 polymorphism was significantly different between oral cancer and control groups who had a smoking habit (p = 0.0198), but was not significant (p = 0.8357) in non-smokers. Consistent with the findings, the C allele frequency was still significantly higher in patients with cancer with a smoking habit than in smoking controls. There was no such difference between the non-smoking groups
Zavras et al.²⁵	239	194	45	ERCC5	PCR	The use of areca nut products, a prevalent habit in southern and eastern Asia that has been linked with higher frequency of epithelial tumor, was significantly higher among subjects with cancer (77.8%) compared with control subjects (15%).
Chuang et al.²⁶	158	101	57	CYP1A1, GSTM1	IHC, ELISA	In smoking groups, the DNA adduct levels were 93.18 ± 81.67 adducts/108 nucleotides, which were significantly higher than in the non-smoking group (0.04 ± 0.33 adducts/108 nucleotides; p < 0.0001). Cigarette smoking may be the major cause of DNA adduct formation in oral tissue
Mondal et al.³¹	124	89	35	GSTM1, GSTT1	PCR	The risk of OSCC is 2.2-fold higher (95%CI: 1.31–3.68; p = 0.002) in tobacco-betel quid chewers, which is one of the main factors for oral cancer and is a common practice in Northeast India. The association between mtDNA copy number and OSCC risk was evident among tobacco-betel quid chewers rather than tobacco-betel quid non-chewers; the interaction between mtDNA copy number and tobacco-betel quid was significant (P = 0.0005). Similar results were observed when cases and controls were classified as tobacco- betel quid chewers and non-chewers based on low and high mtDNA copy number: the tobacco-betel quid chewers with the low mtDNA copy number had a 3.54-fold increased risk of OSCC (95%CI: 1.59–7.87).
Anil et al.⁸	100	72	28	PARP-1 variants	PCR	A strong association was observed with PARP1 SNP rs1136410 homozygous genotype “C/C” polymorphism in OSCC patients with smoking habit. OSCC patients with smoking habit showed nearly 12-fold higher risk compared to healthy individuals with “CC” genotype of SNP rs1136410 (p = 0.01923; OR: 12.615; 95%CI: 0.682-233.37). Similarly, variant allele C imposed 2.547-fold increased risk of OSCC in patients with smoking habit (p = 0.01617; OR: 0.547; 95%CI: 1.165–5.568). PARP1 SNP rs3219090, which didn’t show any association with OSCC cancer in overall study, showed no association with OSCC in patients with smoking habit. No significant association was observed in case of interaction of smoking status with any genotypes in PARP1 SNP rs3219090 and rs1136410 for OSCC risk in non-smokers.
Nigam et al.³²	72	46	26	XPC	PCR	Frequency of smokers among cases was significantly higher than in healthy controls. However, compared to oral cancer subjects, the proportion of nonsmokers was significantly higher among the healthy control group (p = 0.001). The result shows that smoking increases the risk of oral cancer by five times (OR 5.03; 95%CI: 2.91–8.69).

Variable	Certainty assessment							Number of individuals		Certainty
Outcome	Studies	Design	Bias	Inconsistency	Indirectness	Imprecision	Publication	Smokers	Non-smokers	Certainty
glutathione S-transferase mu 1 (GSTM1)	3	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	105/192 (54.7%)	38/61 (62.3%)	⨁◯◯◯
glutathione S-transferase mu 1 (GSTM1)	3	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	105/192 (54.7%)	38/61 (62.3%)	Very low
glutathione S-transferase theta 1 (GSTT1)	2	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	18/47 (38.3%)	17/46 (37.0%)	⨁◯◯◯
glutathione S-transferase theta 1 (GSTT1)	2	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	18/47 (38.3%)	17/46 (37.0%)	Very low
hydrolase proteins (Ku70 and Ku80)	2	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	47/56 (83.9%)	21/24 (87.5%)	⨁◯◯◯
hydrolase proteins (Ku70 and Ku80)	2	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	47/56 (83.9%)	21/24 (87.5%)	Very low
transferase proteins (NAT14, NAT110, GSTM1, GSTT1, and GSTM)	6	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	265/413 (64.2%)	71/124 (57.3%)	⨁◯◯◯
	6	observational studies	not serious	not serious	serious^a	serious^b	publication bias strongly suspected^c	265/413 (64.2%)	71/124 (57.3%)	Very low

[1] Corresponding Author: Vanessa Fátima Bernardes E-mail: bernardesvf@icb.ufmg.br

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Effects of tobacco on the DNA of smokers and non-smokers affected by OSCC: systematic review and meta-analysis

Abstract