乙醇脱氢酶2基因多态和饮酒习惯与肝癌易感性

目的：研究乙醇脱氢酶2（ADH2）基因多态性和饮酒习惯与肝癌的易感性关系。方法：在肝癌高发区江苏省泰兴市采用病例对照研究方法，病例为确诊的原发性肝癌（207例）新发病例，对照（207例）按1：1配对，选取与病例同性别、年龄相差不超过2岁，与病例居住同村或同一街道的非肿瘤居民，调查研究对象的饮酒习惯等因素，并采用聚合酶链反应-限制性片段长度多态性（PCR-PFLP）方法检测了他们的ADH2基因型。结果：1）病例组及对照组中ADH2＊1／＊1、ADH2＊1／＊2、ADH2＊2／＊2的基因型分布频度分别为10．14％、63．77％、26．09％及12．56％、46．86％、40．58％。与携带ADH2＊1／＊1基因型者相比，无论是携带ADH2＊2／＊2（OR=0．8，95％CI=0．39～1．64）或ADH2＊1／＊2（OR=1．68，95％CI=0．86～3．32）基因型者，患肝癌的危险性均无明显增加。2）携带ADH2＊1／＊2或ADH2＊2／＊2基因型饮酒者与携带ADH2＊1／＊1基因型的不饮者相比，惠肝癌的风险差异无统计学意义（OR=0．997，95％CI=0．296～3．354）。3）在调整了年龄、性别、HBsAg、肝硬化和血吸虫病因素后，结合饮酒习惯将前者与后者相比，若开始饮酒年龄〈25岁、饮酒量〉3kg／月、饮酒年数〉23年和饮酒总量（千克年）〉3时，前者惠肝癌的危险性分别是后者的1．88（95％CI=0．53～6．70）、1.35（95％CI=0．36～5．04）、1.46（95％CI=0．41～5．17）和1．46（95％CI=0．39～5．47）倍，差异均无统计学意义。结论：ADH2基因多态与肝癌易感性间无明显联系。     

乙醛脱氢酶2基因多态性和饮酒习惯与肝癌、胃癌、食管癌的易感性

目的：研究乙醛脱氢酶2（ALDH2）基因多态性和饮酒习惯与肝癌、胃癌和食管癌的易感性。方法：88例肝癌以性别、年龄和居住地与对照按1：1配对；104例胃癌和98例食管癌选择同一组（235例）非肿瘤居民为对照，用PCR－RFLP方法检测研究对象的ALDH2基因型。结果：肝癌、胃癌、食管癌病例与对照组中ALDH2变异基因型携带者分别占36．36％和35．23％、47．12％和45．36％、32．65％和45．36％，差异无显著性。在携带ALDH2变异基因型的饮酒人中，每月饮酒总量＞3kg者发生肝癌的危险性是≤3kg者的7．2倍，并存在显著的剂量效应关系。在携带ALDH2谷氨酸纯合型的男性中，饮酒者发生癌的危险性是不饮酒者的2．69倍。结论：该研究揭示了携带ALDH2变异基因型者大量饮酒将显著增加患肝癌的危险性。     

乙醇脱氢酶2基因多态和饮酒习惯与肝癌易感性

目的:研究乙醇脱氢酶2(ADH2)基因多 态性和饮酒习惯与肝癌的易感性关系。方法:在 肝癌高发区江苏省泰兴市采用病例对照研究方 法,病例为确诊的原发性肝癌(207例)新发病例, 对照(207例)按1∶1配对,选取与病例同性别、年 龄相差不超过2岁,与病例居住同村或同一街道 的非肿瘤居民,调查研究对象的饮酒习惯等因素, 并采用聚合酶链反应 限制性片段长度多态性 (PCR PFLP)方法检测了他们的ADH2基因型。 结果:1)病例组及对照组中ADH2 1/ 1、 ADH2 1/ 2、ADH2 2/ 2的基因型分布频 度分别为10.14%、63.77%、26.09%及12.56%、 46.86%、40.58%。与携带ADH2 1/ 1基因型 者相比,无论是携带ADH2 2/ 2(OR=0.8, 95%CI=0.39～1.64)或ADH2 1/ 2(OR= 1.68,95%CI=0.86～3.32)基因型者,患肝癌的危 险性均无明显增加。2)携带ADH2 1/ 2或 ADH2 2/ 2基因型饮酒者与携带ADH2 1/ 1基因型的不饮者相比,患肝癌的风险差异 无统计学意义(OR=0.997,95%CI=0.296～ 3.354)。3)在调整了年龄、性别、HBsAg、肝硬化和 血吸虫病因素后,结合饮酒习惯将前者与后者相 比,若开始饮酒年龄<25岁、饮酒量>3kg/月、饮 酒年数>23年和饮酒总量(千克年)>3时,前者 患肝癌的危险性分别是后者的1.88(95%CI=0.53 ～6.70)、1.35(95%CI     

乙醇脱氢酶2和乙醛脱氢酶2基因多态性及饮酒习惯与直肠癌易感性的研究

目的:研究乙醇脱氢酶2(ADH2)、乙醛脱氢酶2(ALDH2)基因多态性与直肠癌易感性的关系。方法:采用病例-对照研究方法,以PCR-DH-PLC检测研究对象的ADH2/ALDH2基因型,比较不同的基因型及生活习惯与直肠癌的关系。结果:1)与不饮酒者相比较,饮酒者患直肠癌的危险性显著增高(OR=2.20,95%CI:1.47～3.28,P=0.000);2)多因素分析结果未发现ADH2、ALDH2各基因型与直肠癌的危险性有关。3)对ADH2、ALDH2基因多态相互作用的分层分析发现,同时携带ADH2A/A和ALDH2G/G基因型者,发生直肠癌的危险性显著增高(性别、年龄和吸烟调整OR=1.82,95%CI:1.07～3.09)。4)在饮酒者中,ADH2A/A、A/G G/G基因型者患直肠癌的调整OR分别为2.56(95%CI:1.38～4.73)和2.10(95%CI:1.15～3.84);ALDH2G/G基因型者发生直肠癌的调整OR为1.82(95%CI:1.07～3.09)。结论:饮酒是直肠癌的危险因素;ADH2A/A与ALDH2G/G基因型在增加直肠癌易感性上有协同作用;ALDH2G/A A/A基因型可减弱饮酒患直肠癌的危险性。     

醇醛脱氢酶基因多态及饮酒习惯与胃癌易感性

[目的]探讨乙醇脱氢酶2（ADH2）和乙醛脱氢酶2（ALDH2）基因多态及饮酒习惯与胃癌易感性的关系。[方法]选取江苏省泰兴市和常熟市382例男性胃癌新发病例作为病例组,同时按同居住地、同性别及年龄±2岁1：1个体匹配抽取对照。收集所有研究对象的饮酒习惯等因素。采用聚合酶链反应（PCR）和变性高效液相色谱法（DHPLC）检测研究对象ADH2和ALDH2基因型。[结果]①病例组与对照组相比,ADH2和ALDH2各基因型分布频度差异均未达统计学意义。（9携带ALDH2G／A或A／A且酒精消耗总量≥2．5kg·年者,患胃癌的OR值分别是携带ALDH2G／G且酒精消耗总量〈2．5kg·年及≥2．5kg·年者的2．53（95％CI：0．86—7．49）和2．46（95％CI：0．90～6．72）倍,亦是携带ALDH2G／A或A／A且酒精消耗总量〈2．5kg·年者的2．72（95％CI：0．89～8-31）倍,但差异均未达到统计学意义（P〉0．05）。②与同时携带ADH2A／A和AI。DH2G／G者相比．同时携带ADH2G／A或G／G和ALDH2G／A或A,A者无论是否饮酒,患胃癌风险均无明显增加,即使当饮酒者的酒精消耗总量≥2．5kg·年亦如此（OR=3．13,95％CI：0．78,12．64）。[结论]本研究未发现ADH2和ALDH2基因多态及饮酒与胃癌易感性有关。     

饮酒增加广西乙醛脱氢酶2 基因变异基因型携带者发生肝癌的危险性

 目的 研究乙醛脱氢酶2（ALDH2）基因多态性与饮酒因素交互作用在广西原发性肝癌发生中的作用。方法 对广西壮族自治区300例肝癌患者和292例正常对照人群进行流行病学调查研究，并用PCR-RFLP方法检测ALDH2基因型。结果 肝癌组和对照组中ALDH2变异基因型携带者分别占50．33％和47．95％（P=0．561）。肝癌人群ALDH2基因型不存在区域和民族差异（P〉0．05）。饮酒频度每周≥3次的ALDH₂²携带者发生肝癌的危险性是饮酒频度每周〈3次的ALDH2＊1携带者的3、34倍（95％CI1．75～6．41）。HBsAg阳性者发生肝癌的危险性明显高于HbsAg阴性者（P〈0．001）。结论 ALDH2基因型不构成壮汉族间肝癌发病差异的基础，频繁饮酒可能是ALDH2基因变异基因型携带者发生肝癌的危险因素。     

饮酒及酒精代谢酶基因多态与食管癌发病风险

[目的]研究饮酒及乙醇脱氢酶2（ADH2）和乙醛脱氢酶2（ALDH2）基因多态性与食管癌发病风险关系。[方法]对江苏省泰兴市221食管癌新发病例和191对照调查饮酒习惯等因素,采用聚合酶链反应（PCR）和变性高效液相色谱法（DHPLC）检测研究对象ADH2和ALDH2基因型。[结果]①携带ALDH2 G/A或A/A饮酒者患食管癌危险性,分别是携带ALDH2G/G饮酒者或不饮酒者的3.08倍和3.05倍（OR=3.08,95%CI：1.65～5.78;OR=3.05,95%CI：1.49～6.25）。②携带ALDH2 G/A或A/A且酒精消耗总量≥2.5kg·年者患食管癌风险是携带ALDH2G/G且酒精消耗总量〈2.5kg·年者12.31倍（OR=12.31,95%CI：3.01～50.37）。③携带ALDH2 G/A或A/A者患食管癌风险,随酒精消耗总量增加呈趋势性显著上升（P=0.023）,当达到≥2.5kg·年时患食管癌风险是不饮酒者的6.58倍。④同时携带ALDH2 G/A或A/A和ADH2 G/A或G/G且酒精消耗总量≥2.5kg·年者患食管癌风险,是同时携带ALDH2G/G和ADH2A/A且酒精消耗总量〈2.5kg·年者（OR=35.59,95%CI：1.86～680.1）。[结论]可将酒精消耗总量达≥2.5kg·年作为显著增加携带ALDH2 G/A或A/A饮酒者患食道癌风险的阈值。对同时携带ALDH2 G/A或A/A和ADH2 G/A或G/G且酒精消耗总量≥2.5kg·年者,劝其戒酒和定期随访对降低食管癌危害十分有益。     

醇醛脱氢酶基因多态和饮酒习惯与肝癌易感性

目的研究乙醇脱氢酶2（ADH2）和乙醛脱氢酶2（ALDH2）基因多态及饮酒习惯与肝癌的易感性。方法对208例原发性肝癌和208例对照调查饮酒习惯,采用RCT-RFLR方法检测ADH2和ALDH2基因型。结果1）病例与对照ADH2和ALDH2基因型分布频率差异均无统计学意义。2）携带AL-DH2^1＊2或ALDH2^2＊2基因型且饮酒总量〉3kg年者,发生肝癌危险性是携带AL-DH211基因型不饮酒者的3.30倍（95%CI=1.24～8.83）;而携带ADH2^1＊2或ADH2^2＊2基因型者且饮酒总量〉3kg年与携带ADH211基因型不饮酒者相比,患肝癌危险性无显著增加。3）携带AL-DH2^1＊2或ALDH2^2＊2同时携带ADH2^1＊2或ADH2^2＊2基因型且饮酒总量〉3kg年者,与携带ALDH2^1＊1同时携带ADH2^1＊1基因型且饮酒总量≤3kg年者相比,患肝癌OR值虽有增加但未达显著性（OR=4.26,95%CI=0.63～29.08）。4）HBsAg阳性并携带ALDH2^1＊2或ALDH2^2＊2基因型且饮酒〉3kg年者,与HBsAg阴性并携带ALDH2^1＊1基因型且饮酒≤3kg年者相比,患肝癌危险升高49.71倍（95%CI=5.51～448.96）。结论大量饮酒和肝癌的关联与ALDH2基因有关,而与ADH2基因无关。     

乙醛脱氢酶2基因多态性和饮酒习惯与肝癌发生的关系研究

目的 :研究当地饮酒习惯与乙醛脱氢酶 2 (ALDH2 )基因的多态性对肝癌危险性的影响。方法 :在江苏泰兴市选择 1999年 9月 1日～ 2 0 0 0年 12月 31日新发肝癌病人 88例 ,按性别、年龄和居住地1∶1配对 ,进行流行病学调查研究 ,并应用PCR RFLP方法检测研究对象的ALDH2基因型。统计分析采用EpiInfo软件包。结果 :饮大量高度酒能显著增加ALDH2变异基因型携带者患肝癌的危险性 (OR=7 2 0 ,χ2 =6 6 5 ,P <0 0 1)。结论 :ALDH2基因多态性和饮酒习惯与肝癌的发生有关。     

p53 protein accumulation, iodine-unstained lesions, and alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 genotypes in Japanese alcoholic men with esophageal dysplasia

Inactive heterozygous aldehyde dehydrogenase-2 (ALDH2(*)1/(*)2) and less-active alcohol dehydrogenase-1B (ADH1B(*)1/(*)1) increase the risk of esophageal cancer in East Asian drinkers, and esophageal cancer multiplicity is strongly associated with ALDH2(*)1/(*)2. p53 alterations are key molecular events in multifocal carcinogenesis in the esophagus. We studied 260 esophageal-cancer free Japanese alcoholics with esophageal dysplasia diagnosed by biopsy of distinct iodine-unstained lesions (DIULs) ≥5mm. The degree of p53 protein accumulation was positively associated with the degree of atypia (p<0.0001) and size (p=0.040) of DIULs and with the presence of multiple DIULs (p=0.070), but not with ALDH2(*)1/(*)2 or ADH1B(*)1/(*)1.     

Relationship between plasma cell-free DNA (cfDNA) and prognosis of TACE for primary hepatocellular carcinoma

BackgroundOur study aims to investigate changes in cell-free DNA (cfDNA) concentration and integrity in primary hepatocellular carcinoma (PHC) patients before and after transcatheter arterial chemoembolization (TACE) treatment and their influence on the evaluation of prognosis of the disease.MethodsA total of 84 PHC patients admitted to the Affiliated Hospital of Nanjing University of Chinese Medicine from December 2016 to December 2017 were included as the study group, while 55 healthy people served as the control group. Plasma cfDNA concentration and integrity were determined using qRT-PCR. The correlation between cfDNA concentration/integrity and clinical characteristics of PHC patients were analyzed. A ROC curve was used to investigate the sensitivity and specificity of cfDNA as detection indices. Univariate and multivariate analyses were used to analyze factors affecting recurrence in PHC patients and compare recurrence-free survival (RFS) of PHC patients with high cfDNA expression and low cfDNA expression.ResultsPlasma cfDNA concentration and integrity were significantly higher in PHC patients before TACE treatment than in healthy people and significantly lower after treatment than before (P<0.05). The cfDNA concentration was significantly correlated with tumor size, lymph node metastasis, TNM stage, and BCLC stage, while cfDNA integrity was significantly correlated with tumor size, TNM stage, and BCLC stage (P<0.05). ROC results showed that the area under the curve (AUC) value of cfDNA concentration was the largest, with an optimal cut-off of 10.51 ng/mL. Multivariate regression analysis for COX showed that the TNM stage, cfDNA concentration, and AFP were independent risk factors that affected PHC patients’ survival.ConclusionsPlasma cfDNA concentration in PHC patients is more sensitive and specific than any other tumor marker. It is an independent risk factor for PHC patients treated with TACE. Therefore, it is hypothesized cfDNA is a potential biomarker for prognostic evaluation of PHC patients treated with TACE.     

肝动脉栓塞化疗联合放疗治疗不能手术的原发性肝癌的临床观察

目的　观察肝动脉栓塞化疗（ＴＡＣＥ）联合直线加速器或全身伽玛刀治疗不能手术的原发性肝癌（ＰＨＣ）的疗效及毒副反应。方法　２００５年７月至２００８年６月,１０８例不能手术的ＰＨＣ患者中５０例行ＴＡＣＥ联合直线加速器放疗（加速器组）,５８例行ＴＡＣＥ联合全身伽玛刀放疗（伽玛刀组）。ＴＡＣＥ灌注化疗药物包括丝裂霉素（ＭＭＣ）１０～２０ｍｇ、氟尿嘧啶（５-ＦＵ）１０００～１５００ｍｇ、表阿霉素（Ｅ-ＡＤＭ）３０～５０ｍｇ,栓塞剂为４０％超液态碘化油５～２０ｍｌ。直线加速器治疗用６ＭＶ Ｘ,９５％等剂量线包绕ＰＴＶ,４０～６０Ｇｙ／１５～２５ｆ,３～５ｆ／周;伽玛刀治疗用月亮神立体定向伽玛射线旋转聚焦全身放射治疗系统（ＬＵＮＡ-ＴＭ-２６０）,４０％ ～６０％等剂量线包绕ＰＴＶ,单次剂量３～６Ｇｙ,３～５ｆ／周,照射总量３０～５０Ｇｙ。联合应用ＴＡＣＥ为１～３个疗程。结果　加速器组及伽玛刀组的中位生存期分别为１４个月和１６个月,中位肿瘤进展时间（ＴＴＰ）分别为７.６个月和８.１个月;２年局部控制率分别为４５.４％和４３.６％（χ^２ ＝０.０２０,Ｐ＝０.８８７）,３年局部控制率分别为３６.５％和３７.９％（χ^２ ＝０.０４０,Ｐ＝０.８４１）;２年生存率分别为４１１％和３９６％（χ^２ ＝０.０２１,Ｐ＝０.８８５）,３年生存率分别为３４.３％和３０.２％（χ^２ ＝０.３６８,Ｐ＝０.５４４）。加速器组中出现１例放射诱发的肝病,伽玛刀组未见相关病例。结论　直线加速器和伽玛刀联合ＴＡＣＥ治疗ＰＨＣ均安全可靠,疗效相当。     

EPHX代谢酶的遗传多态性和肝癌易感性的相关性研究

目的探讨环氧化物水解酶(EPHX)和谷胱甘肽转硫酶(GST)基因型及血清黄曲霉素B1(AFB1)加成物含量和肝癌易感性的相关性研究.方法以3对肝癌高发家族(62例)和相对应的非癌对照家族成员(58例)为研究对象,分别采用放射免疫法,PCR法测定所有成员血清中AFB1-白蛋白加成物量、HBsAg及血细胞的EPHX,GSTT1,GSTM1的基因型.结果1.高发家族成员有41.9%(26/62)血清AFB1-白蛋白加成物含量高于所有被测成员的中位值,而对照家族仅有15.5%(9/58)高于中位值,二组之间呈显著性差异(P＜0.001).2.在对照家族和高发家族AFB1-白蛋白含量低于中位值的人群中GSTM1,GSTT1,EPHX基因型的分布未见明显的差异;而AFB1-白蛋白含量高于中位值的高发家族人群,其EPHX 113编码位突变型百分率明显高于对照家族(P＜0.001),但GSTT1,GSTM1的基因型的分布无显著性差异.结论EPHX基因113编密子的突变与机体AFB暴露后形成的加成物的含量增加有关,从而可以推断与个体对黄曲霉素的敏感性和肝癌的易感性相关.     

Alcohol dehydrogenase-2 and aldehyde dehydrogenase-2 genotypes, alcohol drinking and the risk of primary hepatocellular carcinoma in a chinese population.

Objective: To investigate the relationship of alcohol dehydrogenase-2 (ADH2) and aldehyde dehydrogenase-2 (ALDH2) genotypes as well as alcohol drinking to the susceptibility of primary hepatocellular carcinoma (HCC). Methods: A case-control study including 208 cases of HCC and 208 controls matched with sex, age and residential area was carried out in Taixing city of Jiangsu province, China. Blood samples were collected and tested for ADH2 and ALDH2 genotypes by PCR-RFLP method. Results: There were no significant differences in the frequency of either ADH2 or ALDH2 genotypes between cases and controls. Compared with no-drinkers possessing ALDH21*1 genotypes, drinkers with ALDH21*2 or ALDH22*2 genotypes and cumulative amount of alcohol consumption >3 (Kg * years) were at a significantly higher risk of developing HCC (OR=3.30, 95%CI: 1.24-8.83). In contrast, there was no significant difference in cancer risk between no-drinkers with ADH21*1 and drinkers with ADH2 1*2 or ADH22*2 genotypes. A dose-dependent positive result was found (P=0.044) between cumulative amount of alcohol consumption and the risk of HCC in individuals carrying ALDH21*2 or ALDH22*2 genotypes. Drinkers with cumulative amount of alcohol consumption >3 (Kg * years) who possessed both inactive ALDH2 (ALDH21*2 or ALDH22*2) and inactive ADH (ADH21*2 or ADH22*2) genotypes were not at a significantly higher risk of HCC (adjusted OR=4.26, 95%CI: 0.63-29.08) compared to no-drinkers possessing ADH21*1 and ALDH21*1 genotypes. Compared with individuals possessing ALDH21*1, with negative HBsAg and cumulative amount of alcohol consumption 3 (Kg * years) had a significantly higher risk of HCC (OR=49.71, 95%CI: 5.51-448.96). Conclusion: These results revealed that it was not ADH2 but ALDH2 polymorphisms that had a significant interaction with heavy alcohol consumption in the development of HCC. This result suggests that to help lower their risk for HCC , persons with ALDH21*2 or ALDH22*2 genotypes should be encouraged to reduce their consumption of alcoholic beverages.     

Influence of alcohol consumption and gene polymorphisms of ADH2 and ALDH2 on hepatocellular carcinoma in a Japanese population

Although alcohol intake as well as hepatitis viruses has been associated with hepatocellular carcinoma (HCC), gene-alcohol interactions on HCC risk remain to be elucidated. We conducted a case-control study to examine whether polymorphisms of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) modified the HCC risk depending on the amount of alcohol intake. ADH2 and ALDH2 genotyping was performed by a duplex polymerase chain reaction with confronting two-pair primers in 209 newly diagnosed HCC cases and 2 different controls [275 hospital controls and 381 patients with chronic liver disease (CLD)]. Multiple logistic regression analyses revealed that heavy drinkers consuming >or=3 "go"s/day of sake (69 g of ethanol/day) showed an increased risk of HCC based on comparison of HCC cases with hospital controls [adjusted odds ratio (OR) = 13.5; 95% confidence interval (CI) 3.3-54.3] or CLD patients (adjusted OR = 7.0; 95% CI 2.5-19.2), whereas the overall risk was not elevated among light to moderate drinkers consuming <3 "go"s/day. Interestingly, light to moderate drinking was associated with an increased risk among those with ALDH2*1/*2 (adjusted OR = 4.5 or 2.0), but not among those with ALDH2*1/*1 (adjusted OR = 0.8 or 1.0; p interaction = 0.03 or 0.13). However, this gene-alcohol interaction was not observed for heavy drinking. Among light to moderate drinkers, people with the combination of ALDH2*1/*2 and ADH2*2/*2 revealed the highest risk of HCC. These findings indicate that the ALDH2 polymorphism may modify HCC risk among light to moderate drinkers.     

Interaction between interleukin-1beta -31T/C gene polymorphism and drinking and smoking habits on the risk of hepatocellular carcinoma among Japanese

The risk of hepatocellular carcinoma (HCC) increases with the severity of hepatic inflammation. Interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha are proinflammatory cytokines with multiple biological effects and may play essential roles in inflammation-linked tumor development. We conducted a case-control study including 209 incident HCC cases and two control groups (275 hospital controls and 381 patients with chronic liver disease [CLD] without HCC) to investigate whether IL-1B and TNF-A gene polymorphisms influence HCC susceptibility with any interaction with alcohol and tobacco. By comparing HCC cases with CLD patients, we found that IL-1B -31T/C polymorphism was associated with HCC risk among never drinkers and current smokers; adjusted odds ratios (and 95% confidence intervals) for C/T and T/T genotypes compared with C/C genotype were 1.70 (0.76-3.77) and 2.46 (1.05-5.76) (P trend=0.03), respectively, among never drinkers, and 1.53 (0.60-3.99) and 2.54 (0.81-7.95) (P trend=0.11), respectively, among current smokers. Similarly, HCC risk associated with heavy alcohol intake and current smoking differed by this polymorphism among CLD patients. IL-1B -31T/C polymorphism may modify HCC risk in relation to alcohol intake or smoking.