Author’s Accepted Manuscript The role of reactive oxygen species (ROS) and cytochrome P-450 2E1 in the generation of carcinogenic etheno-DNA adducts Kirsten Linhart, Helmut Bartsch, Helmut K. Seitz www.elsevier.com

PII: DOI: Reference:

S2213-2317(14)00099-8 http://dx.doi.org/10.1016/j.redox.2014.08.009 REDOX203

To appear in: Redox Biology Received date: 4 August 2014 Revised date: 19 August 2014 Accepted date: 25 August 2014 Cite this article as: Kirsten Linhart, Helmut Bartsch and Helmut K. Seitz, The role of reactive oxygen species (ROS) and cytochrome P-450 2E1 in the generation of carcinogenic etheno-DNA adducts, Redox Biology, http://dx.doi.org/10.1016/j.redox.2014.08.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Manuscript

1 2 3

7

The role of reactive oxygen species (ROS) and cytochrome P-450 2E1 in the generation of carcinogenic etheno-DNA adducts

8 9 10 11 12 13 14 15 16

Kirsten Linhart, Helmut Bartsch and Helmut K. Seitz,

4 5 6

17 18 19 20 21 22 23 24 25 26 27 28

Centre of Alcohol Research, University of Heidelberg, Heidelberg, Germany Department of Medicine (Gastroenterology & Hepatology), Salem Medical Centre, Heidelberg, Germany & Division of Toxicology and Cancer Risk Factors, German Cancer Research Centre (DKFZ), Heidelberg, Germany

29 30 31 32

Correspondence:

33

Helmut K. Seitz, MD, AGAF

34

Professor of Medicine, Gastroenterology and Alcohol Research

35

University of Heidelberg

36

Department of Medicine,

37

Salem Medical Centre, Heidelberg, Germany

38

Tel.: 0049-6221-483 200; Fax: 0049-6221-483 494

39

E-Mail: [email protected]

1

Abstract:

2

Exocyclic etheno-DNA adducts are mutagenic and carcinogenic and are formed by

3

the reaction of lipidperoxidation (LPO) products such as 4-hydoxynonenal or

4

malondialdehyde with DNA bases. LPO products are generated either via

5

inflammation driven oxidative stress or via the induction of cytochrome P-450 2E1

6

(CYP2E1). In the liver CYP2E1 is induced by various compounds including free fatty

7

acids, acetone and ethanol. Increased levels of CYP2E1 and thus, oxidative stress

8

are observed in the liver of patients with non-alcoholic steatohepatitis (NASH) as well

9

as in the chronic alcoholic. In addition, chronic ethanol ingestion also increases

10

CYP2E1 in the mucosa of the oesophagus and colon. In all these tissues CYP2E1

11

correlates significantly with the levels of carcinogenic etheno-DNA adducts. In

12

contrast, in patients with non-alcoholic steatohepatitis (NASH) hepatic etheno-DNA

13

adducts do not correlate with CYP2E1 indicating that in NASH etheno-DNA adducts

14

formation is predominately driven by inflammation rather than by CYP2E1 induction.

15

Since etheno-DNA adducts are strong mutagens producing various types of base

16

pair substitution mutations as well as other types of genetic damage, it is strongly

17

believed that they are involved in ethanol mediated carcinogenesis primarily driven

18

by the induction of CYP2E1.

19 20

Keywords:

21

Cytochrome P450-2E1, etheno-DNA adducts, lipidperoxidation products, ethanol,

22

carcinogenesis, non-alcoholic fatty liver disease

23 24 25

1. Introduction

26

Oxidative stress is an important mechanism in the pathogenesis of many diseases

27

including cancer. The generation of reactive oxygen species (ROS) with consecutive

28

DNA damage is an initial step in carcinogenesis induced by inflammatory processes.

29

During inflammation ROS is generated among others through various cytokines, but

30

also through other mechanisms such as the induction of cytochrome P4502E1

31

(CYP2E1) as demonstrated following chronic alcohol consumption (1,2). This review

32

will focus on the effect of alcohol on CYP2E1 and its role in ROS formation, but major

33

emphasis will be led on the generation of carcinogenic exocyclic etheno-DNA

34

adducts as a consequence of the reaction between lipidperoxidation products

2

1

generated by ROS and DNA bases following ethanol administration in vitro and in

2

vivo. It will be shown that these etheno-DNA adducts following chronic ethanol

3

consumption

4

carcinogenesis in the liver and in other tissues.

are

of

major

importance

with

respect

to

ethanol

mediated

5 6

2. Inflammation, Oxidative Stress and DNA damage

7

Chronic inflammation induced by various agents including viruses and bacteria is

8

associated with an increased cancer risk due to tissue damage and genetic instability

9

(3-7). Oxidative stress with the generation of ROS may occur in chronic infection and

10

inflammation primarily due to the generation of nitric oxide (NO), superoxide anion

11

(O2.-) and other ROSs by macrophages and neutrophils that infiltrate the inflamed

12

tissue (8,9).

13

Activated inflammatory cells in various tissues including the liver in turn induce

14

oxidant generating enzymes such as NADPH oxidase, inducible nitric oxide

15

synthetase (iNOS), xanthine oxidase (XO) and myeloperoxidase (MPO) (10,11). In

16

such conditions ROS and reactive nitrogen species (RNS) are generated. As a

17

consequence ROS and RNS can damage DNA, RNA, lipids and proteins through

18

nitration and oxidation resulting in an increased mutation load (10,11).

19

Furthermore, cytokines are released in inflammatory tissues which not only activate

20

the above mentioned enzymes to create ROS and RNS, which also activate NFκB a

21

nuclear transcription factor which among others stimulates cyclooxygenase 2(COX2),

22

lipoxygenase (LOX), and iNOS (4,10-12). Upregulation of iNOS, COX2, and LOX

23

results also in an overproduction of ROS and RNS (10).

24

iNOS catalyzes nitric oxide (NO) generation which reacts with oxygen to produce

25

N2O3 a strong nitrosating compound which deaminates DNA bases and react with

26

secondary amines to form N-nitrosoamines which are highly carcinogenic (10).

27

Another reaction with O2 leads to peroxynitrite with the formation of 8-nitroguanine.

28

Peroxynitrite also results in single strand breakage of DNA (7,10).

29

COX2 catalyze the conversion of arachidonic acid (AA) to prostaglandins is inducible

30

by various factors including NFκB, cytokines and tumour promoters and may

31

influence apoptosis, angiogenesis, tumour invasion, but also the generation of

32

oxidative stress (12,13). An upregulation of COX2 has been shown in familial

33

adenomatous polyposis (FAP) and in the Apc Min mouse model which resembles

3

1

FAP and this was associated with a highly significant increase in various etheno-DNA

2

lesions (13-16).

3

LOX metabolizes AA to hydroxyeicosatetraenoic acids (HETEs) or leukotrienes. It

4

has been shown in mouse skin carcinogenesis that LOX isoenzymes are

5

overactivated and some of the metabolites cause chromosomal damage which was

6

found to be inhibited by LOX inhibitors (17,18).

7

Thus, all the factors mentioned above lead to the generation of ROS and RNS with

8

consequent lipid peroxidation and the production of lipidperoxidation products such

9

as

4-hydoxynonenal

(4HNE),

4-hydoxyhydroperoxy-2-nonenal

(HPNE)

and

10

malondialdehyde (MDA) (Fig1). These lipidperoxidation products react with DNA

11

either directly or through bifunctional intermediates to form various promutagenic

12

exocyclic etheno-DNA adducts. Some major types are depicted in Fig.2.

13

LPO products derived from y-linoleic acid, including HNE, a major LPO product and

14

its electrophilic epoxy-, hydroperoxy-, and oxo-enal intermediates react with the DNA

15

bases A, C, and G to yield inter alia the unsubstituted etheno-DNA adducts 1,N6-

16

etheno-2’-deoxyadenosine (εdA), 3, N4-etheno-2’-deoxycytidine (εdC), 1,N2-etheno-

17

2’-deoxyguanosine (1,N2εdG), and N2,3-etheno-2’-deoxyguanosine (N2,3εdG). In

18

addition, also substituted base adducts are formed such as HNE-dG carrying a fatty

19

acid chain residue (Fig. 2). 2,N4-etheno-5-methyl-2’-deoxycytidine (ε5mdC), an

20

endogenous, hitherto unknown LPO-derived adduct was identified in the DNA of

21

human tissue which could play a role in epigenetic mechanisms of carcinogenesis.

22

(10,19-27).

23

In addition, DNA can also be modified directly by ROS and RNS to 8-nitro-dG and 8-

24

Oxo-dG (28). All of these DNA changes have been detected in human specimens

25

(29-34).

26 27 28

3. The Importance of Etheno DNA Adducts in Carcinogenesis

29

Exocyclic etheno-DNA adducts exhibit strong mutagenic properties producing various

30

types of base pair substitution mutations and other types of genetic damage in all

31

organisms tested so far (35,36). εdA can lead to ATGC transition and ATTA and

32

ATCG transversions (37,38). εdC can cause CGAT transversions and CGTA

33

transition (39,40), and N2,3εdG can lead to GCAT transition (40). Incorporation of a

4

1

single εdA in either DNA strand of HeLa cells showed a similar miscoding frequency

2

and was more mutagenic than 8-oxo-dG (41).

3

Some etheno adducts are poorly repaired in some tissues and cells supporting their

4

biological relevance (42). Strong support that etheno-DNA adducts play a causal role

5

in the initiation and progression of liver carcinogenesis comes from the formation of

6

εdA and εdC in vivo by the human liver carcinogen vinyl chloride (43) and by the

7

potent multiorgan, multispecies carcinogen urethane via their reactive epoxy-

8

intermediates (44). The biological importance of etheno-DNA adducts is further

9

stressed as they are preferentially formed in codon 249 of TP53 (which encodes

10

p53), leading to a mutation that renders cells more resistant to apoptosis and

11

provides them some growth advantage (45).

12

LPO-derived reactive products and their macromolecular interactions have been so

13

far characterized primarily by in vitro studies, making it difficult, to pinpoint the main

14

precursors and pathways involved in the generation of cancer-relevant DNA damage

15

in human in vivo. For this reason earlier studies analyzed in human specimens εdA

16

and εdC as maker lesions for several other exocyclic adducts that could be formed

17

with DNA in vivo, for which sensitive detection methods were not yet available.

18

Using ultrasensitive and specific detection methods (46), two miscoding etheno-DNA

19

adducts εdA and εdC and also ε5mdC were unequivocally identified in humans.

20

Samples were collected from “at-risk” patients affected by chronic inflammatory

21

processes, persistent viral infections, iron storage- and alcohol-related diseases or

22

exposed to inherited / acquired cancer risk factors. Adduct levels increased 10-100-

23

fold progressively in human cancer-prone organs including liver, bile duct,

24

oesophagus, colon and pancreas. Consistent results were also observed in rodent

25

tumour models, that mimick human disease (for review 47). Taken together these

26

data incriminate LPO-derived adducts as strongly mutagenic cancer-causing lesions.

27 28 29

4. Alcohol and Oxidative stress

30

Chronic ethanol consumption may results in the development of alcoholic liver

31

disease (ALD) and cancer of various sites including the liver and the upper

32

aerodigestive tract (2,48). One mechanism by which alcohol exerts its deleterious

33

effects is the generation of ROS. As already pointed out the formation of ROS such

34

as superoxide anion (O2-) and hydrogen peroxide (H2O2) causes oxidative injury

5

1

(1,2). ROS as well as acetaldehyde, the first metabolite of ethanol oxidation, both

2

activate NFκB an important transcription factor involved in carcinogenesis (48).

3

Inflammation driven oxidative stress including activated hepatic macrophages as

4

observed in alcoholic hepatitis (AH) is predominantly responsible for the generation

5

of ROS in AH (49). Also hepatic iron overload as observed in the alcoholic increases

6

ROS (50,51). Furthermore, ethanol also results in the increase of iNOS with an

7

increased production of nitric oxide and the generation of the highly reactive

8

peroxynitrite (ONOO-) (52).

9

In addition, several enzyme systems are capable to produce ROS including CYP2E1

10

as part of the microsomal ethanol oxidizing system (MEOS) which metabolizes

11

ethanol to acetaldehyde in the presence of oxygen and NADPH (53,54). This system

12

is of major importance, since it can be induced by chronic consumption of ethanol. It

13

has been shown that CYP2E1 induction occurs already at a daily ethanol dose of 40

14

grams and already at 1 week of consumption, which is further enhanced with time.

15

However, an interindividual intensity of CP2E1 increase has been observed. Some

16

individuals react to alcohol consumption with a striking CYP2E1 induction, while

17

others reveal only a weak induction of CYP2E1 (55). It is noteworthy that CYP2E1

18

induction by ethanol may also depend on dietary factors since medium chain

19

triglycerides diminish CYP2E1 induction as compared to the application of long chain

20

triglycerides in animal experiments (56).

21

CYP2E1 has a high rate of NADPH oxidase activity, resulting in the generation of

22

large quantities of O2-, H2O2 and hydroxyethyl radicals (1,2,57). Thus, CYP2E1

23

dependent microsomal ethanol oxidation produces ROS leading to lipidperoxidation

24

with the generation of lipidperoxidation products such as 4-hydroxynonenal (4-HNE)

25

and malondialdehyde (MDA) (1,2).

26

The role of CYP2E1 in the formation of ROS, in the progression of ALD and in

27

ethanol mediated carcinogenesis has been clearly demonstrated (2,58-62). Thus, the

28

severity of ALD was significantly enhanced in CYP2E1 overexpressing mice (63),

29

and reduced in CYP2E1 knock-out mice (62). When chlormethiazole (CMZ), a strong

30

and specific CYP2E1 inhibitor was given in addition to an ethanol containing diet

31

which induces ALD a significant reduction of ROS and RNS was noted in the liver of

32

these animals (62) associated with a striking improvement of the liver disease (64).

33

Subsequently, oxidized DNA lesions have been found to be lower in CYP2E1 knock-

34

out mice as compared to wild type mice following chronic alcohol administration (65).

6

1

3.1. CYP2E1 induction and etheno-DNA adduct generation in the liver

2

3.1.1. Effect of Ethanol

3

We have recently investigated the effect of CYP2E1 on lipidperoxidation products

4

and etheno-DNA lesions in HepG2 cells overexpressing CYP2E1, and in humans

5

with alcoholic liver disease (66). When HepG2 cells overexpressing CYP2E1 were

6

incubated with increasing concentrations of ethanol up to 50mM, an increasing load

7

of εdA and εdC could be detected as compared to control cells. This was not only a

8

concentration dependent, but also a time dependent process. However, when 20µM

9

CMZ were added to the cell culture a highly significant inhibition of the generation of

10

etheno-DNA adducts was observed (66).

11

Since increased levels of εdA adducts have been observed in the hepatic nuclei of

12

patients with ALD (30), we extended our experiments and studied liver biopsies from

13

alcoholic patients with various severities of ALD by using immunohistology for the

14

detection of CYP2E1 and etheno-DNA adducts. Again there was a significant

15

correlation between CYP2E1, the lipidperoxidation product 4-HNE and εdA, as well

16

as εdC (66).

17

Most recently, we have investigated CYP2E1 and exocyclic etheno-DNA adducts in a

18

large cohort study of 97 alcoholics with non-cirrhotic ALD. All patients were liver

19

biopsied, histologically evaluated and CYP2E1 as well as etheno-DNA adducts were

20

immunohistologically determined. As a result a strong significant correlation between

21

CYp2E1 and εdA (p=0.0001) has been observed (Seitz, personal observation).

22 23 24

3.1.2. Non-Alcoholic Fatty Liver Disease (NAFLD)

25

CYP2E1 is not only induced by chronic ethanol ingestion, but also in NAFLD (67-69)

26

possibly by free fatty acids and acetone (54). Although, this induction is less

27

pronounced as in ALD, it also has severe consequences with respect to the

28

generation of oxidative stress. Indeed, inflammation driven oxidative stress may be

29

an important mechanism in the progression of NAFLD (70). We therefore determined

30

CYP2E1 as well as εdA in liver biopsies from patients with pure non-alcoholic fatty

31

liver and patients with NASH using immunohistology. εdA was detected in a broad

32

range of intensity and correlated significantly with the severity of inflammation, but

33

not with CYP2E1 (Linhart and Seitz, personal communication).

7

1

NAFLD also is an increasing health problem in children (71-73). As reported recently,

2

oxidative stress as measured by the hepatic expression of 8-hydroxy-2-

3

deoxyguanosine (8-OHG), serum protein carbonyls, and circulating antibody against

4

malondialdehyde adducted human serum albumin has been frequently found in

5

children with NAFLD and was also found to be associated with an increased severity

6

of steatohepatitis (74). Therefore, we determined εdA, and CYP2E1 in liver biopsies

7

of children with NASH. In these studies we also could show for the first time that not

8

only CYP2E1 was found to be increased, but also that εdA occurs. In a few of these

9

children at an age below 15 years and the diagnosis of diabetes mellitus a striking

10

load of εdA was found in the nuclei of their hepatocytes (75). In contrast to ALD these

11

adducts did not significantly correlate with CYP2E1, but rather with the state of

12

inflammation. Thus, inflammatory driven ROS production may be predominant to

13

explain εdA formation in patients with NAFLD.

14

In animal experiments, the progression of NASH is influenced by the concomitant

15

administration of ethanol (76,77). This has been shown in dietary induced NASH,

16

which could be due among others to oxidative, nitrosative, and mitochondrial stress,

17

as well as increased inflammation and cellular apoptosis (76,77).

18

Furthermore, in the Zucker rat, a leptin deficiency and insulin resistance genetic

19

NASH model the administration of ethanol not only increased CYP2E1, but also εdA

20

in a linear way (66).

21

The fact that ethanol consumption in patients with NASH enhances oxidative stress

22

possibly predominantly by a further increase in CYP2E1 associated with the

23

generation of highly carcinogenic etheno-DNA lesions may of special interest in the

24

context that patients with NASH have significant higher HCC risk and develop HCC in

25

a much shorter time frame when they consume alcohol even at social levels (78).

26 27 28

3.2. CYP2E1 induction and etheno-DNA adduct generation in the oesophagus

29

and in the colorectal mucosa.

30

Chronic alcohol consumption is a major risk factor for esophageal cancer (2,79).

31

Various mechanisms may mediate carcinogenesis including the genotoxic effect of

32

acetaldehyde and oxidative stress (2,79-81). As discussed above for the liver,

33

ethanol may also exert its carcinogenic effect in other tissues among others via the

34

induction of CYP2E1 and the generation of carcinogenic etheno-DNA adducts.

8

1

Therefore, we investigated if such effects can also be observed in the human

2

esophagus (82). We studied esophageal biopsies of 37 patients with upper

3

aerodigestive tract cancer and heavy alcohol consumption of more than 100 grams

4

on average per day as well as 16 controls without tumors (12 teetotalers and 4

5

subjects with a maximum of 25 g ethanol/day). CYP2E1, etheno-DNA adducts and

6

Ki67 as a marker for cell proliferation were determined immunohistologically in the

7

esophageal mucosa adjacent to the tumour. Chronic alcohol ingestion resulted in a

8

significant induction of CYP2E1 which correlated with the amount of alcohol

9

consumed. Furthermore, a significant correlation between CYP2E1 and the

10

generation of the carcinogenic exocyclic etheno-DNA adducts εdA and εdC was

11

observed. Etheno-DNA adducts also correlated significantly with cell proliferation,

12

which was especially enhanced in patients who both drank and smoked. The results

13

showed clearly again a correlation between CYP2E1 and etheno-DNA adducts. In

14

contrast to the liver this induction correlated significantly with the amount of ethanol

15

ingested (82).

16

More recently, we also investigated immunohistologically the effect of ethanol on

17

colorectal CYP2E1 and etheno-DNA adducts in colorectal biopsies from 31 alcoholics

18

and 15 non drinking controls (Linhart and Seitz, personal communication). Again we

19

found a significant correlation between the two parameters. It is interesting that

20

chronic

21

hyperproliferation of the colorectal mucosa with an extension of the proliferative

22

compartment towards the lumen of the crypt, which is a first step in carcinogenesis of

23

this tissue (83). A similar observation was made in patients with heavy alcohol

24

consumption (84). The administration of vitamin E, a radical scavenger, however,

25

reduced the proliferative rate significantly emphasizing indirectly that most likely

26

oxidative stress induced by CYP2E1 may be responsible for this regenerative

27

behavior (85).

ethanol

consumption

using

a Lieber

DeCarli

diet

resulted

in

a

28 29 30

3.3. CYP2E1 and experimental hepatocarcinogenesis

31

It has been believed for a long time that ethanol by itself is not a carcinogen rather

32

than a co-carcinogen or a tumour promoter. However, meanwhile various animal

33

studies have demonstrated that the administration of ethanol alone without any

34

chemical carcinogen can result in tumours of the liver (86), the upper aerodigestive

9

1

tract (87), the mammary gland (88) and the intestine (89). Besides the fact that DNA

2

lesions induced either by the binding of acetaldehyde to DNA (2,48) or by the

3

reaction of DNA with ROS do occur during chronic ethanol consumption, the role of

4

CYP2E1 in this process has not intensively investigated.

5

In a series of experiments we used an animal model in which a small amount of

6

diethylnitrosamine

7

hepatocarcinogenesis (90,91). CYP2E1, inflammatory proteins, cell proliferation,

8

protein bound 4-HNE, etheno-DNA adducts as well as 8-hydroxy-2’-deoxyguanosine

9

(8-OHdG), retinoid concentrations and hepatic carcinogenesis were examined.

10

Chronic ethanol ingestion for 1 month resulted in increased CYP2E1 levels and an

11

increased nuclear accumulation of NFκB protein. In addition, TNFα expression was

12

also enhanced associated with increased cyclin D1 expression and p-GST positive

13

altered hepatic foci. All these changes were significantly inhibited by the concomitant

14

administration of CMZ. Following 10 months of ethanol feeding hepatocellular

15

adenoma were detected in ethanol fed rats only, but not in control rats. The

16

administration of CMZ inhibited completely the formation of hepatic adenomas. In

17

addition, 8-OHdG formation was found to be significantly increased after alcohol and

18

almost normalized with CMZ. Although, etheno-DNA adduct formation increased

19

following ethanol ingestion and decreased with CMZ, this effect was not significant.

20

More recently, Tsuchishima and co-workers produced hepatocellular carcinoma in

21

mice without any additional insult. This process was significantly associated with the

22

expression of CYP2E1 (92).

(20mg/kg

b.wt.)

was

administered

once

to

initiate

23 24 25

4. Summary

26

The most important mechanism associated with oxidative stress and the generation

27

of ROS is chronic inflammation. During inflammation cytokines are liberated resulting

28

in the activation of oxidant generation enzymes such as NADPH oxidase, and NFκB

29

with the activation of LOX, Cox-2 and iNOS finally leading to the formation of ROS. In

30

addition, ROS can also be generated through CYP2E1 which is induced by chronic

31

alcohol consumption as well as in NASH where free fatty acids as well as acetone

32

(mostly in diabetics) induce CYP2E1.ROS leads to lipidperoxidation with the

33

occurrence of lipidperoxidation products such as 4-HNE and MDA. Both compounds

34

can bind to DNA forming highly carcinogenic etheno-DNA adducts. In a series of

10

1

experiments we could show that a significant correlation exists between CYP2E1

2

levels and etheno-DNA adduct formation in cell culture, animal experiments and

3

biopsies from patients with ALD. Since in NASH the inflammatory process

4

predominates as compared to the induction of CYP2E1 the etheno-DNA adduct

5

levels do not correlate with CYP2E1 but rather with the intensity of the inflammatory

6

process.

7 8

5. Conclusion

9

Cell culture and animal experiments as well as clinical biopsy studies in patients with

10

ALD emphasize an important role of CYP2E1 in alcohol mediated carcinogenesis in

11

the liver, but also in other tissues. In addition, CYP2E1 seems to be a driving force in

12

the progression of ALD. Inhibition of CYP2E1 by a nontoxic inhibitor may be a

13

successful approach in the treatment of ALD and alcohol mediated carcinogenesis.

14 15 16

Acknowledgements: The authors want to thank Ms Grönebaum for typing the

17

manuscript. Original studies were supported by the Dietmar Hopp Foundation and by

18

the Manfred Lautenschläger Foundation.

19 20

11

1

References:

2

1

3

2006;65:278-290.

4

2

5

carcinogenesis. Nat Rev Cancer 2007;7:599-612.

6

3

7

26;420:860-867.

8

4

9

Cancer 2003;3:276-285.

Albano E. Alcohol, oxidative stress and free radical damage. Proc Nutr Soc

Seitz

HK,

Stickel

F.

Molecular

mechanisms

of

alcohol-mediated

Coussens LM, Werb Z. Inflammation and cancer. Nature 2002 Dec 19-

Hussain SP, Hofseth LJ, Harris CC. Radical causes of cancer. Nat Rev

10

5

Kawanishi S, Hiraku Y. Oxidative and nitrative DNA damage as biomarker for

11

carcinogenesis with special reference to inflammation. Antioxid Redox Signal

12

2006;8:1047-1058.

13

6

14

Nature 2008;454:436-444.

15

7

16

cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res

17

1994;305:253-264.

18

8

19

infections to chronic inflammation and cancer. Cell 2006;124:823-835.

20

9

21

inflammation: reconciling chemical mechanisms and biological fates. Int J Cancer

22

2011;128:1999-2009.

Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation.

Ohshima H, Bartsch H. Chronic infections and inflammatory processes as

Karin M, Lawrence T, Nizet V. Innate immunity gone awry: linking microbial

Lonkar P, Dedon PC. Reactive species and DNA damage in chronic

12

1

10

Bartsch H, Nair J. Chronic inflammation and oxidative stress in the genesis

2

and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair.

3

Langenbecks Arch Surg 2006;391:499-510.

4

11

5

development and progression. Nat Rev Immunol 2005;5:749-759.

6

12

7

Cyclooxygenases in cancer: progress and perspective. Cancer Lett 2004;215:1-20.

8

13

9

and prostaglandin H synthase-2. Cell 1996;87:783-786.

Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer

Zha S, Yegnasubramanian V, Nelson WG, Isaacs WB, De Marzo AM.

Prescott SM, White RL. Self-promotion? Intimate connections between APC

10

14

Schmid K, Nair J, Winde G, Velic I, Bartsch H. Increased levels of

11

promutagenic etheno-DNA adducts in colonic polyps of FAP patients. Int J Cancer

12

2000;87:1-4.

13

15

14

Beauchamp RD, et al. Elevated cyclooxygenase-2 levels in Min mouse adenomas.

15

Gastroenterology 1996;111:1134-1140.

16

16

17

mediated DNA-adduct formation in min mice. J Biol Chem 2006;281:10127-10133.

18

17

19

unscheduled eicosanoid signaling and tumor development: cancer chemoprevention

20

by inhibitors of arachidonic acid metabolism. Toxicology 2000;153:11-26.

Williams CS, Luongo C, Radhika A, Zhang T, Lamps LW, Nanney LB,

Williams MV, Lee SH, Pollack M, Blair IA. Endogenous lipid hydroperoxide-

Marks F, Muller-Decker K, Furstenberger G. A causal relationship between

13

1

18

Nair J, Furstenberger G, Burger F, Marks F, Bartsch H. Promutagenic etheno-

2

DNA adducts in multistage mouse skin carcinogenesis: correlation with lipoxygenase-

3

catalyzed arachidonic acid metabolism. Chem Res Toxicol 2000;13:703-709.

4

19

5

deoxyguanosine with the aldehydes trans-4-hydroxy-2-hexenal and trans-4-hydroxy-

6

2-nonenal in vitro. Cancer Res 1986;46:5682-5686.

7

20

8

source for the formation of exocyclic DNA adducts. Carcinogenesis 1996;17:2105-

9

2111.

Winter CK, Segall HJ, Haddon WF. Formation of cyclic adducts of

Chung FL, Chen HJ, Nath RG. Lipid peroxidation as a potential endogenous

10

21

El Ghissassi F, Barbin A, Nair J, et al. Formation of 1,N6-ethenoadenine and

11

3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases. Chem

12

Res Toxicol 1995;8:278-283.

13

22

14

products with DNA. A review. Mutat Res 1988;195:137-149.

15

23

16

2-nonenal from the autoxidation of polyunsaturated fatty acids. Free Radic Biol Med

17

1990;8:541-543.

18

24

19

283:15545-15549.

20

25

21

adducts by 2,3-epoxy-4-hydroxynonanal. Cancer Res 1991;51:137-143.

Vaca CE, Wilhelm J, Harms-Ringdahl M. Interaction of lipid peroxidation

Pryor WA, Porter NA. Suggested mechanisms for the production of 4-hydroxy-

Blair IA. DNA adducts with lipid peroxidation products. J Biol Chem 2008;

Sodum RS, Chung FL. Stereoselective formation of in vitro nucleic acid

14

1

26

Nair U, Bartsch H, Nair J. Lipid peroxidation-induced DNA damage in cancer-

2

prone inflammatory diseases: a review of published adduct types and levels in

3

humans. Free Radic Biol Med 2007;43:1109-1120.

4

27.

5

source for the formation of exocyclic DNA adducts. Carcinogenesis 1996;17:2105-

6

2111.

7

28.

8

carcinogenesis. In: Cancer and Inflammation Mechanisms (eds. Y Hiraku, S

9

Kawanishi, H Ohshima) 2014, pp 41-59.

Chung FL, Chen HJ, Nath RG. Lipid peroxidation as a potential endogenous

Hiraku Y, Kawanishi S. Role of nitrative DNA damage in inflammation related

10

29

Eberle G, Barbin A, Laib RJ, et al. 1,N6-etheno-2'-deoxyadenosine and 3,N4-

11

etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of

12

rats exposed to vinyl chloride. Carcinogenesis 1989;10:209-12.

13

30

14

ethenodeoxyadenosine in nuclei of human liver affected by diseases predisposing to

15

hepato-carcinogenesis. Carcinogenesis 2004;25:1027-1031.

16

31

17

tissues, white blood cells, and urine by ultrasensitive (32)P-postlabeling and

18

immunohistochemistry. Methods Mol Biol 2011;682:189-205.

19

32

20

ethenodeoxycytosine in liver DNA from humans and untreated rodents detected by

21

immunoaffinity/32P-postlabeling. Carcinogenesis 1995;16:613-617.

Frank A, Seitz HK, Bartsch H, et al. Immunohistochemical detection of 1,N6-

Nair J, Nair UJ, Sun X, et al. Quantifying etheno-DNA adducts in human

Nair J, Barbin A, Guichard Y, et al. 1,N6-ethenodeoxyadenosine and 3,N4-

15

1

33

Nair J, Godschalk RW, Nair U, et al. Identification of 3,N(4)-etheno-5-methyl-

2

2’-deoxycytidine in human DNA: a new modified nucleoside which may perturb

3

genome methylation. Chem Res Toxicol 2012;25:162-169.

4

34

5

peroxidation-derived DNA damage in patients with cancer-prone liver diseases. Mutat

6

Res 2010;683:23-8.

7

35

8

tumor mutation spectra. Mutat Res 2000;462:55-69.

9

36.

Nair J, Srivatanakul P, Haas C, et al. High urinary excretion of lipid

Barbin A. Etheno-adduct-forming chemicals: from mutagenicity testing to

Bartsch H, Barbin A, Marion MJ, Nair J, Guichard Y. Formation, detection, and

10

role in carcinogenesis of ethenobases in DNA. Drug Metab Rev 1994;26:349-371.

11

37.

12

and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-

13

ethenoadenine,

14

Biochemistry 1993;32:12793-12801.

15

38.

16

mutagenic in mammalian cells. Biochemistry 1996;35:11487-11492.

17

39.

18

sequence analysis of mutational hot spots. Frequency and specificity of mutations

19

induced by a site-specific ethenocytosine in M13 viral DNA. Biochemistry

20

1993;32:4105-4111.

21

40.

22

DNA adducts: marked differences between Escherichia coli and simian kidney cells.

23

Proc Natl Acad Sci U S A 1994;91:11899-11903.

Basu AK, Wood ML, Niedernhofer LJ, Ramos LA, Essigmann JM. Mutagenic

3,N4-ethenocytosine,

and

4-amino-5-(imidazol-2-yl)imidazole.

Pandya GA, Moriya M. 1,N6-ethenodeoxyadenosine, a DNA adduct highly

Palejwala VA, Rzepka RW, Simha D, Humayun MZ. Quantitative multiplex

Moriya M, Zhang W, Johnson F, Grollman AP. Mutagenic potency of exocyclic

16

1

41.

Levine RL, Yang IY, Hossain M, Pandya GA, Grollman AP, Moriya M.

2

Mutagenesis induced by a single 1,N6-ethenodeoxyadenosine adduct in human

3

cells. Cancer Res 2000;60:4098-4104.

4

42

5

formed in DNA of vinyl chloride-exposed rats are highly persistent in liver.

6

Carcinogenesis 1992;13:727-729.

7

43

8

chloride DNA derivative N2,3-ethenoguanine produces G----A transitions in

9

Escherichia coli. Proc Natl Acad Sci U S A 1991;88:9974-9978.

Swenberg JA, Fedtke N, Ciroussel F, Barbin A, Bartsch H. Etheno adducts

Cheng KC, Preston BD, Cahill DS, Dosanjh MK, Singer B, Loeb LA. The vinyl

10

44

Miller JA, Miller EC. The metabolic activation and nucleic acid adducts of

11

naturally-occurring carcinogens: recent results with ethyl carbamate and the spice

12

flavors safrole and estragole. Br J Cancer 1983;48:1-15

13

45.

14

lipid peroxidation product, trans-4-hydroxy-2-nonenal, preferentially forms DNA

15

adducts at codon 249 of human p53 gene, a unique mutational hotspot in

16

hepatocellular carcinoma. Carcinogenesis 2002;23:1781-1789.

17

46.

18

adducts in human tissues, white blood cells, and urine by ultrasensitive (32)P-

19

postlabeling and immunohistochemistry. Methods Mol Biol 2010;682:189-205

20

47.

21

inflammation-related carcinogenesis. In: Hiraku Y, Kawanishi S, Ohshima H, eds.

22

Cancer and inflammation mechanisms

Hu W, Feng Z, Eveleigh J, Iyer G, Pan J, Amin S, Chung FL, et al. The major

Nair J, Nair UJ, Sun X, Wang Y, Arab K, Bartsch H. Quantifying etheno-DNA

Bartsch H, Nair J: Lipid peroxidation-derived DNA adduts and the role in

17

1

Chemical, Biological and Clinical Aspects. Hoboken, New Jersey: John Wiley &

2

Sons, 2014; 61-74.

3

48

4

with special emphasis on alcohol and oxidative stress. Biol Chem 2006;387:349-360.

5

49

6

21.

7

50.

8

Breizthaupt-Heinlein, Dick TP, Seitz HK, Muckenthaler MU, Mueller S. Sustained

9

submicromolar H2O2 induced hepcidin via signal transducer and activator of

Seitz HK, Stickel F. Risk factors and mechanisms of hepatocarcinogenesis

Bautista AP. Neutrophilic infiltration in alcoholic hepatitis. Alcohol 2002;27:17-

Millonig G, Ganzleben I, Peccerella T, Casanovas G, Brodziak-Jarosz L,

10

transcriptor 3 (STAT3). J Biol Chem 2912;287:37472-82.

11

51

12

oxidative damage to DNA in nonalcoholic steatohepatitis. Cancer Epidemiol

13

Biomarkers Prev 2009;18:424-432.

14

52

15

lipopolysaccharide administration. Alcohol Clin Exp Res 1996;20:1065-1070.

16

53.

17

vitro characteristrics and adaptive properties in vivo. J Biol Chem 245(10):2505-2512.

18

54.

Lieber CS (2004) CYP2E1: from ASH to NASH. Hepatol Res 28(1):1-11.

19

55.

Oneta CM, Lieber CS, Li JJ, Rüttimann S, Schmid B, Lattmann J, Rosman A,

20

Seitz HK (2002) Dynamics of cytochrome P4502E1 activity in man: induction by

21

ethanol and disappearance during withdrawal phase. J. Hepatol 36(1):47-52.

Fujita N, Miyachi H, Tanaka H, et al. Iron overload is associated with hepatic

Chamulitrat W, Spitzer JJ. Nitric oxide and liver injury in alcohol-fed rats after

Lieber CS, DeCarli LM (1970) Hepatic microsomal ethanol oxidizing system: In

18

1

56

Lieber CS, Cao Q, DeCarli LM, Leo MA, Mak KM, Ponomarenko A, Ren C, et

2

al. Role of medium-chain triglycerides in the alcohol-mediated cytochrome P450 2E1

3

induction of mitochondria. Alcohol Clin Exp Res 2007;31:1660-1668.

4

57.

5

Role of cytochrome P-4502E1-dependent formation of hydroxyethyl free radical in the

6

development of liver damage in rats intragastrically fed with etrhanol. Hepatology

7

1996;23:155-163.

8

58

9

fatty liver disease. J Hep 2013, 58: 395–398.

Albano E, Clot P, Morimoto M, Tomasi A, Ingelman-Sundberg M, French SW.

Leung TM, Nieto N. CYP2E1 and oxidant stress in alcoholic and non-alcoholic

10

59

Dey A, Cederbaum AI. Induction of cytochrome P450 2E1 promotes liver injury

11

in ob/ob mice. Hepatology 2007;45:1355-1365.

12

60.

13

hepatotoxicity caused by Fas agonistic Jo2 antibody in mice. Hepatology

14

2005;42:400-410.

15

61.

16

mediated toxicity in HepG2 cells. Hepatology 37(6): 1395-1404. .

17

62.

18

contributes to ethanol-induced fatty liver in mice. Hepatology 47(5):1483-1494.

19

63.

20

transgenic mouse and initial evaluation of alcoholic liver damage. Hepatology

21

2002;36:122-134.

Wang X, Lu Y, Cederbaum AI. Induction of cytochrome P450 2E1 increases

Perez MJ, Cederbaum AI (2003) Proteasome inhibition potentiates CYP2E1-

Lu Y, Zhuge J, Wang X, Bai J, Cederbaum AI (2008) Cytochrome P450 2E1

Morgan K, French SW, Morgan TR. Production of a cytochrome P450 2E1

19

1

64.

Gouillon Z, Lucas D, Li J, Hagbjork AL, French BA, Fu P, Fang C, et al.

2

Inhibition of ethanol-induced liver disease in the intragastric feeding rat model by

3

chlormethiazole. Proc Soc Exp Biol Med 2000;224:302-308.

4

65.

5

L, et al. Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide

6

phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent

7

liver. Hepatology 2005;41:336-344.

8

66.

9

HK (2009): Ethanol-induced cytochrome P-4502E1 causes carcinogenic etheno-DNA

Bradford BU, Kono H, Isayama F, Kosyk O, Wheeler MD, Akiyama TE, Bleye

Wang Y, Millonig G, Nair J, Patsenker E, Stickel F, Mueller S, Bartsch H, Seitz

10

lesions in alcoholic liver disease. Hepatology 50(2): 453-461.

11

67.

12

cytochrome P-4502E1 is increased in patients with nonalcoholic steatohepatitis.

13

Hepatology 1998;27:128-133.

14

68

15

2E1 (CYP2E1) in the development of high fat-induced non-alcoholic steatohepatitis. J

16

Hepatol 2012;57:860-866.

17

69

18

activity in nondiabetic patients with nonalcoholic steatohepatitis. Hepatology

19

2003;37:544-50.

20

70

21

Gastroenterol 2012;47:215-225.

22

71

23

problem? J Hepatol 2007;46:1133-1142.

Weltman M, Farrell G, Hall P, Ingelman-Sundberg M, Liddle C. Hepatic

Abdelmegeed MA, Banerjee A, Yoo SH, et al. Critical role of cytochrome P450

Chalasani N, Gorski JC, Asghar MS, et al. Hepatic cytochrome P450 2E1

Fujii H, Kawada N. Inflammation and fibrogenesis in steatohepatitis. J

Roberts EA. Pediatric nonalcoholic fatty liver disease (NAFLD): a "growing"

20

1

72

Ruiz-Extremera A, Carazo A, Salmeron A, et al. Factors associated with

2

hepatic steatosis in obese children and adolescents. J Pediatr Gastroenterol Nutr

3

2011;53:196-201.

4

73

5

steatohepatitis in obese children. Gastroenterol Nurs;2011;34:434-437.

6

74

7

Oxidative stress parameters in paediatric non-alcoholic fatty liver disease. Int J Mol

8

Med, 2010;26:471-476.

9

75

Lerret SM, Garcia-Rodriguez L, Skelton J, et al. Predictors of nonalcoholic

Nobili V, Parola M, Alisi A, Marra F, Piemonte F, Mombello C, Sutti S, et al.

Qin H, Teufel U, Engelmann G , et al. Detection of hepatic highly carcinogenic,

10

exocyclic etheno-DNA-adducts in patients with alcoholic and non-alcoholic fatty liver

11

disease and in children with non-alcoholic steatohepatitis. J Hepatol, 2012, 56

12

(Suppl. 2), S520

13

76

14

Synergistic steatohepatitis by moderate obesity and alcohol in mice despite

15

increased adiponectin and p-AMPK. J Hepatol 2011;55:673-682.

16

77.

17

aggravates high fat diet induced steatohepatitis in rats. Alcoholism ClinExp Res

18

34(3): 567-573.

19

78

20

hepatocellular carcinoma in patients with nonalcoholic steatohepatitis. Hepatology

21

2010;51:1972-1978.

22

79.

23

Cogliano V and the Working Group: Willett WC, Miller AB, Rehm J, Cai L, Bloomfield

Xu J, Lai KK, Verlinsky A, Lugea A, French SW, Cooper MP, Ji C, et al.

Wang Y, Seitz HK, Wang XD (2010) Moderate alcohol consumption

Ascha MS, Hanouneh IA, Lopez R, et al. The incidence and risk factors of

Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Bouvard V, Altieri A,

21

1

K, Eriksson P, Benichou J, Lachenmaier D, Seitz HK , LaVecchia C, Kono S,

2

Yokoyama A, Cho S, Gricuite LL, Weiderpass E, Marques M, Rehn-Mendoza N,

3

Gmel G, Allen N, Beral V, Anderson LM, Beland FA, Brooks PJ, Crabb DW, Gapstur

4

SM, Rusyn I, Zhang ZF (2007) Carcinogenicity of alcoholic beverages. The Lancet

5

Oncology 8(4):192-293.

6

80

7

Increased cancer risk in heavy drinkers with the alcohol dehydrogenase 1C*1 allele,

8

possibly due to salivary acetaldehyde. Gut 2004;53:871-876.

9

81

Visapaa JP, Gotte K, Benesova M, Li J, Homann N, Conradt C, Inoue H, et al.

Homann N, Konig IR, Marks M, Benesova M, Stickel F, Millonig G, Mueller S,

10

et al. Alcohol and colorectal cancer: the role of alcohol dehydrogenase 1C

11

polymorphism. Alcohol Clin Exp Res 2009;33:551-556.

12

82.

13

(2011) Ethanol-mediated carcinogenesis in the human esophagus implicates

14

Cytochrome P-4502E1 induction and the generation of carcinogenic DNA-lesions. Int

15

J Cancer 128(3):533-40.

16

83.

17

TJ, et al. Enhancement of ethanol induced rectal mucosal hyper regeneration with

18

age in F344 rats. Gut 1994;35:1102-1106.

19

84.

20

FX, et al. Increased rectal cell proliferation following alcohol abuse. Gut 2001;49:418-

21

422.

Millonig G, Bernhardt F, Wang Y, Mueller S, Homann N, Bartsch H, Seitz HK

Simanowski UA, Suter P, Russell RM, Heller M, Waldherr R, Ward R, Peters

Simanowski UA, Homann N, Knuhl M, Arce L, Waldherr R, Conradt C, Bosch

22

1

85

Vincon P, Wunderer J, Simanowski UA, Koll M, Preedy VR, Peters TJ, Werner

2

J, et al. Inhibition of alcohol-associated colonic hyperregeneration by alpha-

3

tocopherol in the rat. Alcohol Clin Exp Res 2003;27:100-106.

4

86

5

Doerge DR. Effect of ethanol on the tumorigenicity of urethane (ethyl carbamate) in

6

B6C3F1 mice. Food Chem Toxicol 2005;43:1-19.

7

87

8

of long-term experimental studies on the carcinogenicity of methyl alcohol and ethyl

9

alcohol in rats. Ann N Y Acad Sci 2002;982:46-69.

Beland FA, Benson RW, Mellick PW, Kovatch RM, Roberts DW, Fang JL,

Soffritti M, Belpoggi F, Cevolani D, Guarino M, Padovani M, Maltoni C. Results

10

88

Watabiki T, Okii Y, Tokiyasu T, Yoshimura S, Yoshida M, Akane A, Shikata N,

11

et al. Long-term ethanol consumption in ICR mice causes mammary tumor in females

12

and liver fibrosis in males. Alcohol Clin Exp Res 2000;24:117S-122S.

13

89

14

promotes intestinal tumorigenesis in the MIN mouse. Multiple intestinal neoplasia.

15

Cancer Epidemiol Biomarkers Prev 2002;11:1499-1502.

16

90

17

hepatic tumorigenesis but impairs normal hepatocyte proliferation in rats. J Nutr

18

2011;141:1049-55.

19

91

20

hepatic

21

Hepatobiliary Surg Nutr 2012;1:5-18.

Roy HK, Gulizia JM, Karolski WJ, Ratashak A, Sorrell MF, Tuma D. Ethanol

Chavez PR, Lian F, Chung J, et al. Long-term ethanol consumption promotes

Ye Q, Lian F, Chavez PR, et al. Cytochrome P450 2E1 inhibition prevents carcinogenesis

induced

by

diethylnitrosamine

in

alcohol-fed

rats.

23

1

92

Tsuchishima M, George J, Shiroeda H, et al. Chronic ingestion of ethanol

2

induces hepatocellular carcinoma in mice without additional hepatic insult. Dig Dis

3

Sci 2013;58:1923-1933

4 5 6 7 8 9

24

1

Figures:

2

Figure 1

3 4 5 6

25

1 2

3 4 5 6 7 8 9

Figure 2

Figure 3

10 11 12 13

26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Figure Legends: Figure 1: Simplified pathophysiology of reactive oxygen species (ROS) and etheno-DNA adduct formation. Inflammation driven cytokine secretion results among others in NFκB activation and in the activation of NADPH oxidase (NADPH-Ox) as well as myeloperoxidase (MPO). NFκB is also activated by acetaldehyde, the first metabolite of ethanol oxidation. NFκB stimulates lipoxigenase (LOX), Cyclooxygenase 2 (COX 2), and inducible nitric oxide synthase (iNOS). As a result ROS and reactive nitrogen species (RNS) are generated, which lead to lipidperoxidation with the occurrence of lipidperoxidation products such as 4-hydroxynonenal (4-HNE), 4-hydroxyhydroperoxy-2-nonenal (HPNE), and malondialdehyde (MDA).These adducts react with DNA bases to form exocyclic etheno/propane-DNA adducts. Chronic alcohol consumption results in the induction of cytochrome P-4502 E1 which is involved in ethanol oxidation through the microsomal ethanol oxidizing pathway. During this reaction ROS is generated without inflammation. Other compounds such as free fatty acids or acetone also induce CYP2E1 which is especially relevant in non alcoholic fatty liver disease (NAFLD), when the liver is loaded with fat and in patients with diabetes mellitus when acetone is generated in the liver.

Figure 2: Generation of various etheno DNA-adducts Deoxyadenine, deoxycytosine, and deoxyguanosine react with lipidperoxidation products to form 1,N6-ethano-2’-deoxyadenosine (εdA), 3,N4-etheno-2’-deoxycytidine (εdC), and 1,N2-etheno-2’-deoxyguanosine (1,N2εdG), N2,3-etheno-2’2 deoxyguanosine (N2,3-εdG), and HNE-derived 1,N -propano-2’-deoxyguanosine adduct (HNE-dG).

Figure 3: Immunohistology of εdA in two liver biopsies from patients with alcoholic liver disease (A) and control patients with a normal liver (B) The brownish color shows εdA. This adduct occurs in the nuclei of the hepatocytes and the percentage of nuclei positive hepatocytes can be counted. A significant load of etheno adducts is observed in ALD, while the control healthy liver reveals background activity only.

27

Highlights (for review)

Highlights, Bullet Point: Cytochrome P-450 2E1 is induced following chronic ethanol ingestion CYP2E1 correlates with carcinogenic etheno-DNA formation CYP2E1 and oxidative stress are important mechanisms in alcohol mediated carcinogenesis in the liver, esophagus and colon In NASH hepatic etheno-DNA adducts occur but possible due to inflammation

Graphical Abstract (for review)

Generation of reactive oxygen species (ROS) either via inflammatory cytokines (predominately in NASH) or via cytochrome P-450 2E1 induction (predominately by ethanol, but also in NASH). ROS lead to lipid peroxidation and lipid peroxidation products result in the formation of carcinogenic etheno- or propano-DNA adducts.