Ethanol-induced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure

Panidz Navasumrit1,3, Timothy H. Ward2, Nicholas J.F. Dodd4 and Peter J. O'Connor1,5

1 Cancer Research Campaign, Section of Genome Damage and Repair and
2 Section of Drug Development and Imaging, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester M20 4BX and
4 Department of Medical Biophysics, University of Manchester, Manchester M13 9PT, UK


    Abstract
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 Abstract
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 Materials and methods
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 References
 
Ethanol was given to male Wistar rats as an acute dose (5 g/kg) or continuously at 5% (w/v) in a liquid diet to provide 36% of the caloric requirement. Free radicals generated in the liver were collected as a stable adduct in bile following the in vivo administration of the spin trapping agent {alpha}-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN; 700 mg/kg). [1-13C]ethanol was used to confirm the formation of the 1-hydroxyethyl radical and to demonstrate that this was ethanol-derived in the case of the single-dose treatment. Free radical production increased up to 1h after the acute dose and then plateaued over the next 30 min. During chronic exposure to ethanol, free radical generation increased significantly after 1 week and then declined again to remain at a low level over the next 2 weeks; this transient increase corresponded closely with the induction of cytochrome P-450 2E1 (CYP 2E1) in response to ethanol feeding. Single-cell electrophoresis was used to investigate effects on DNA. After an acute dose of ethanol, the frequency of single-strand breaks increased from 1 h to peak at 6 h but then declined again to control values by 12 h. During the chronic exposure, an increase in the frequency of DNA breaks was seen at 3 days, reached a peak at 1 week and then decreased slowly over the next 5 weeks. The effects of antioxidants on these parameters was investigated after an acute dose of ethanol. Pre-treatment with vitamin C (400 mg/kg, i.p., daily for 5 days) or vitamin E (100 mg/kg, i.p., for 5 days) prior to the administration of ethanol (5 g/kg) inhibited generation of the 1-hydroxyethyl–POBN adduct by 30 and 50%, respectively, and both agents prevented the increased frequency of DNA single-strand breaks caused by ethanol. The significance of the temporal coincidence of changes in the above parameters in response to ethanol is discussed.

Abbreviations: BSA, bovine serum albumin; CYP, cytochrome P450; EGTA, ethylene glycol-bis(2-amino ethyl ether)N,N'-tetraacetic acid; ESR, electron spin resonance; MEOS, microsomal ethanol oxidizing system; POBN, {alpha}-(4-pyridyl-1-oxide)-N-tert-butylnitrone; ROS, reactive oxygen species.


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As interest has increased in the possible roles of oxidative stress and the generation of reactive radicals in the effects of ethanol-induced liver damage, a variety of mechanisms whereby ethanol can influence free radical formation have been studied.

Several groups have focused on the generation of reactive oxygen species (ROS) by the ethanol-inducible protein cytochrome P450 2E1 (CYP 2E1). Microsomal CYP 2E1 from ethanol-treated rats or a human hepatoma-derived cell line (HepG2) have a high capacity to generate reactive oxygen intermediates, such as superoxide radicals (O2.–) and hydrogen peroxide (H2O2) (1,2). These reactive oxygen intermediates subsequently lead to the formation of 1-hydroxyethyl radicals (CH3.CHOH) via a Fenton or a Haber–Weiss reaction arising secondarily to the iron-catalysed formation of hydroxyl radicals (OH.) (1,3,4). These reactions are, however, insensitive to scavengers of hydroxyl radicals; therefore, the generation of the 1-hydroxyethyl radical may arise via the direct catalytic formation of an ethanol radical by abstraction of a hydrogen atom from ethanol at the active site of CYP (5).

Elevated levels of reactive oxyradicals caused by ethanol may also arise as a consequence of an increase in the NADH/NAD+ redox ratio or as a result of the release of iron (6), which has also been associated with an increased cellular NADH/NAD+ ratio (7). For example, an enhanced release of iron from ferritin, a major storage form of iron within cells, resulted from the elevated levels of ethanol-induced CYP 2E1 which increased the generation of O2.– and was effective in stimulating lipid peroxidation in the presence of NADPH and ethanol-treated microsomes (8,9).

Kupffer and endothelial cells are activated after recovery from an acute ethanol `binge' and produce significant amounts of O2.– (10); when Kupffer cells were isolated from ethanol-fed rats they showed a significantly increased production of ROS and lipid peroxidation, compared with those from pair-fed controls (11). Conversely, the destruction of Kupffer cells by chronic treatment with gadolinium chloride decreased the signal intensity of 1-hydroxyethyl radicals in the bile of chronic ethanol-treated rats (3).

Aerobic organisms are normally protected by a defence system against oxidative damage which is induced by reactive radicals. The antioxidant system comprises various compounds with different functions. These include enzymes (e.g. catalase, glutathione peroxidase, glutathione transferase, DT diaphorase and superoxide dismutase) and the sequestration of metals (e.g. iron, copper and hence by chelating agents), all of which suppress the generation of free radicals. In addition, hydrophilic and lipophilic compounds (e.g. vitamin C and vitamin E) which can act by scavenging or suppressing the generation of free radicals, are also important for control of intracellular levels. Vitamin C is a water soluble antioxidant which protects cells from oxidative stress by scavenging free radicals. It has a very low reduction potential and can therefore, inactivate potentially highly damaging radicals, including, OH. and lipid peroxyl radicals (LOO.). {alpha}-Tocopherol is biologically the most active form of vitamin E and is the major lipid-soluble antioxidant present in mammalian cells and blood (12,13). It acts as an antioxidant, primarily by scavenging active oxygen radicals which would otherwise initiate and/or propagate chain reactions and attack substrates such as lipids, proteins, sugars and DNA. {alpha}-Tocopherol is also a potent peroxyl radical scavenger and thereby can prevent the propagation of free radical damage in biological membranes (1416).

A number of investigations have revealed that vitamin C and E levels are decreased by exposure to ethanol. For example, a decrease in the level of vitamin C in rat testis was observed after ethanol administration (17). In healthy volunteers, pre-treatment with vitamin C (1 g daily for 3 days) decreased alcohol toxicity (mediated by circulating acetaldehyde) after drinking 84 g of ethanol (18). Chronic ethanol administration caused an increase in the level of ascorbyl radicals in the bile of alcohol-treated rats (19) and the ascorbyl radical has been proposed as a marker of oxidative stress (20). In the case of vitamin E, chronic ethanol intake leads to a significant decrease in the content of this vitamin in the mitochondrial and microsomal fractions of rat hepatocytochromes, Kupffer cells (11) and blood cells (21,22). A reduced level of vitamin E in serum was also observed in alcoholics with or without liver disease (23) and in the livers of patients with alcoholic liver cirrhosis (24).

The combination of an increased flux of oxygen radicals and the subsequent loss of cellular redox homeostasis can generate various DNA lesions. These include DNA strand breaks, base modifications (e.g. 8-hydroxyguanine and thymine glycol) as well as degradation products of deoxyribose (25). Recent research has suggested that ethanol may exert its cell toxicity via DNA damage, possibly via the generation of ROS arising from microsomal NADPH-dependent electron transfer and the oxidation of the ethanol metabolite, acetaldehyde (26). There was a significant increase in the number of in vivo single-strand breaks in the DNA of rat brain cells after an acute dose of ethanol (27); ethanol combined with acetaldehyde induced cleavage of DNA in rat hepatocytes (28) and ethanol-induced DNA fragmentation and cell death was also observed in mouse thymocytes (29). Ethanol alone, however, did not induce in vitro DNA strand breaks in human lymphocytes, whereas acetaldehyde induced both single- and double-strand breaks (30), indicating that ethanol may induce DNA strand breaks via its primary metabolite. The damaging effect of acetaldehyde may be mediated by the formation of DNA cross-links (31) and acetaldehyde–protein adducts (32); removal and repair of DNA cross-links may then lead to the generation of DNA strand breaks.

Free radicals can be detected using spin trapping agents and they have been used previously to demonstrate the increased generation of 1-hydroxyethyl radical both in vitro (33) and in vivo (34). Using these procedures (34), we have followed the effects of acute and chronic doses of ethanol on the generation of free radicals and we have further attempted to correlate free radical generation with the evolution of DNA strand breaks using single electrophoresis. Results of these studies indicate that ethanol-induced DNA damage is mediated by free radicals and that both can be significantly inhibited by prior treatment with antioxidants.


    Materials and methods
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 Materials and methods
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Chemicals
[1-13C]ethanol was purchased from ICN Biochemicals Inc., OH. All other chemicals were obtained from Sigma Chemical Company (Poole, Dorset, UK).

Animals and their treatments
Male outbred Wistar rats (130–150 g) were obtained from the breeding unit at the Paterson Institute. For acute ethanol treatments, ethanol (5 g/kg; 25% w/v) was given to animals by gavage and the same volume of vehicle was given to the controls. The chronic ethanol-treated animals were fed a liquid diet containing ethanol (5%; w/v) to provide 36% of total energy requirement throughout the study and control rats were pair-fed with an isocaloric liquid diet, both prepared according to Lieber and DeCarli (35). For antioxidant pre-treatments, animals were given vitamin C (L-ascorbic acid, 400 mg/kg) or vitamin E (DL-{alpha}-tocopherol acetate, 100 mg/kg) daily by i.p. injection for 5 days prior to ethanol treatment; the comparable amount of vehicles (water or corn oil) were given to non-pre-treated groups corresponding to vitamin C or E treatments, respectively.

In vivo determination of 1-hydroxyethyl radicals
Bile sample collection.
Control or ethanol-treated Wistar rats were anesthetized with a combination of Ketamin (100 mg/kg) and Xylazine (1 mg/kg) by i.p. injection. Bile ducts and femoral veins were canulated with polyethylene tubing (0.58 mm i.d.; 0.63 mm o.d.). The spin trapping agent, {alpha}-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), was infused slowly through the canulated femoral vein to deliver a dose of 700 mg/kg (34) and bile was collected into 1.5 ml tubes containing 30 µl of 30 mM dipyridyl and 300 mM bathocuproine disulfonic acid to prevent ex vivo formation of free radicals. All samples were frozen immediately on dry ice and kept at –80°C until analysis.

Electron spin resonance spectroscopy (ESR) analysis.
The samples were thawed, warmed to room temperature, then transferred to a flat quartz tube (aqueous cell) and placed in the cavity of a Bruker ECS 106 X-band ESR spectrometer. The ESR conditions were: gain, 1x106; modulation amplitude, 1.0 G; modulation frequency, 100 kHz; and microwave power, 20 mW. Multiple scans were accumulated to provide a definitive ESR spectrum for analysis. The spectrum was then characterized and the intensity of the 1-hydroxyethyl–POBN adduct was measured.

In vitro generation of 1-hydroxyethyl radicals.
A reaction mixture containing 1 mM H2O2 and 20 mM ethanol or [1-13C]ethanol in the presence of 10 mM POBN was subjected to UV irradiation for ~2 min. The ESR spectrum of the 1-hydroxyethyl–POBN adduct was analysed using the same conditions as above.

In vivo detection of DNA strand breaks by single-cell electrophoresis
Single-cell suspensions.
Liver was removed immediately after the animal was killed by stunning followed by cervical dislocation. One lobe was perfused with 0.05 mM EDTA in Hanks buffer with 1% BSA pH 7.4 at 37°C followed by a digestion buffer containing 0.05% (w/v) collagenase and 1% (w/v) BSA in Hanks buffer pH 7.4 at 37°C. A small piece of perfused liver (~100 mg) was transferred to ice-cold Hanks buffer containing 1% (w/v) BSA and 0.05% (w/v) collagenase and gently disrupted and filtered through two cell strainers of 100 and 50 µm, respectively.

Comet analysis.
Cell suspensions were prepared for comet analysis as described previously (36). Briefly, the embedded cells were lysed in 1 l of ice cold lysis buffer (2.5 M NaCl, 100 mM EDTA and 10 mM Tris, pH 10.5) containing 1% (v/v) Triton X-100 and 1% (v/v) DMSO for 1 h in a subdued light and were then washed four times with 1 l of distilled water for 15 min each.

The slides were transferred to an electrophoresis tank and submerged in 1 l of alkali solution (50 mM NaOH and 1 mM EDTA, pH 12.5) for 45 min in order to unwind the DNA. Electrophoresis was subsequently performed at 0.6 V/cm for 25 min. After electrophoresis, the slides were neutralized with 0.5 M Tris–HCl pH 7.5 for 10 min followed by PBS for 10 min and then were dried overnight at room temperature. Following rehydration in distilled water for 30 min, the slides were stained with propidium iodide (2.5 µg/ml) for 30 min then washed with distilled water for 1 h. Slides were then analysed immediately or stored dry at room temperature until analysis.

Comet analysis was carried out using a Zeiss epifluoresence microscope equipped with an intensified solid-state CDD camera and image analysis system. The slide was illuminated with green light from a 50 W mercury lamp with an excitation of 580 nm and a barrier filter of 590 nm; individual comets were viewed at 250x magnification. Twenty-five cells were analysed randomly for each slide and duplicate slides were analysed for each sample. DNA strand breaks were quantified using the parameter `Comet Distant Moment' which is defined as: Dxpercentage DNA in the comet tail; D is the distance between the means of the head and tail distributions (37). Under these experimental conditions, using a 137Cs source (0.4 Gy/min), 10 Gy X-irradiation yields a comet distance moment of 36 ± 2.3.

Cell viability testing
Single cell suspensions (100 µl) were mixed with 800 µl Hanks buffer and 100 µl 3% Trypan blue and placed on ice for ~5 min. Cell mixtures (~20 µl) were transferred to a haemocytometer for determination of cell viability. The cell viability of the liver tissue preparations used in this study was ~90%.

Statistical evaluation
Differences between the means of two groups were tested by the Student's t-test; statistical significance was indicated when the P-value was <0.05.


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Acute ethanol treatment
Generation of 1-hydroxyethyl radicals.
Acute ethanol treatment generates 1-hydroxyethyl radicals that can be detected in bile as an adduct of POBN. The ESR spectrum of the 1-hydroxyethyl–POBN adduct showed six lines arising from hyperfine splittings of aN = 15.6 G and aH = 2.5 G (Figure 1AGo). These values were similar to those reported previously for the 1-hydroxyethyl radical adduct of POBN in aqueous solution (3,34). The signal appeared rapidly in all bile samples from ethanol-treated rats after POBN injection and the signal intensity increased with time. This signal was not detected in untreated animals as shown in Figure 1BGo.




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Fig. 1. Effect of acute ethanol treatment on hepatic 1-hydroxyethyl radical generation. A single oral dose of ethanol (5 g/kg) was administered to rats (~200 g body wt); water was given to control animals. After 30 min, the bile duct was canulated, POBN was infused and bile samples were collected for 10 min periods commencing 5, 35 and 55 min after POBN infusion. The spectrum of the 1-hydroxyethyl–POBN adduct was determined by ESR analysis (A) and the intensity of the peak of POBN adducts was measured (B); each data point is the mean ± SE from four animals.

 
Confirmation that the 1-hydroxyethyl adduct so formed was ethanol derived, was obtained by administering [1-13C]ethanol to rats. The ESR spectrum of the [1-13C]ethanol/POBN adduct in bile gave a 12 line spectrum (aN = 15.6 G, aH = 2.5 G and a13C = 4.1 G), resulting from the additional splitting due to 13C in the spin adducts (Figure 2BGo). Interference by ascorbyl radicals was also detected as the center doublet signal with a hyperfine splitting of aH = 1.8 G (Figure 2BGo). Comparable results were obtained following in vitro generation of the 1-hydroxyethyl–POBN adduct by UV photolysis of H2O2 using either 20 mM ethanol or 20 mM [1-13C]ethanol (Figure 2C and DGo).



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Fig. 2. ESR spectra of the 1-hydroxyethyl–POBN adduct obtained after acute ethanol treatment in vivo or after UV irradiation in vitro. In vivo: 30 min after treatment of rats (~200g body wt) with ethanol (5 g/kg, i.g.) (A) or [1-13C]ethanol (B), bile samples were obtained for analysis as detailed in Figure 1Go. In vitro: 1 mM H2O2 and 20 mM ethanol (C) or [1-13C]ethanol (D) were subjected to UV radiation for 2 min in the presence of 10 mM POBN. *, Doublet signal of the ascorbyl radical.

 
Ethanol-induced DNA strand breaks.
A single oral dose of ethanol (5 g/kg) produced strand breaks in rat liver DNA. The profile (Figure 3Go), shows that the frequency of DNA strand breaks in liver from ethanol-treated rats was increased significantly 1 h after ethanol treatment; it reached a peak after 6 h and then returned to the control level at 12 h post-treatment.



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Fig. 3. Strand breaks in hepatic DNA after acute ethanol treatment. Wistar rats (~200 g body wt) were treated with single oral doses of ethanol (5 g/kg) or given a comparable volume of water. The incidence of DNA strand breaks in the livers of control or ethanol-treated rats was determined at 1, 3, 6 and 12 h after ethanol treatment by Comet assay as described in Materials and methods; each data point is the mean ± SE from four animals. * and **, significantly different from control value at P < 0.05 or P < 0.01, respectively.

 
Chronic ethanol treatment
Generation of free radicals.
The effect of short- and long-term low-level exposure to ethanol on the generation of free radicals was studied from 3 days up to 6 weeks in rats maintained on a liquid diet containing 5% (w/v) ethanol.

The results showed that the signal intensity of the POBN adducts generated in bile was increased significantly in the ethanol-fed animals after 1 week of feeding (Figure 4Go). However, the POBN adduct was also observed in bile from pair-fed controls and this component could have arisen from the interaction of endogenous lipid and POBN, since the adduct of the lipid radical and POBN gives rise to an ESR spectrum which resembles that of the 1-hydroxyethyl–POBN adduct (38).



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Fig. 4. Time-course effects of ethanol (5%; w/v) administered in a liquid diet on hepatic free radical generation. Bile samples from pair-fed controls or ethanol-fed rats were collected at various intervals over the 6 week period. At each interval, 90 min after POBN injection, samples were collected for a period of 10 min and analysed as described in Figure 1Go; each data point is the mean ± SE from three to nine animals. *, Significantly different from the corresponding control value at P < 0.05.

 
Ascorbyl radicals were also observed in bile from both ethanol-treated animals and pair-fed controls.

Ethanol induced DNA strand breaks.
Ethanol-induced DNA strand breaks were also observed in rats fed a liquid diet containing 5% (w/v) ethanol for a period of 6 weeks.

Figure 5Go shows that the ethanol-containing liquid diet induced significant levels of hepatic DNA damage at 1 week and throughout the 6 weeks of feeding when compared with the isocaloric pair-fed controls. The incidence of DNA strand breaks in ethanol-treated animals showed a maximum after 1 week and then gradually decreased over the next 5 weeks. The frequency of DNA strand breaks also increased in the liver of control animals, but this occurred slowly and progressively throughout the study period, so that after 4 and 6 weeks of feeding the value was significantly greater than that seen at 3 days.



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Fig. 5. Time-course effect of ethanol (5%; w/v) administered in a liquid diet on the induction of hepatic DNA strand breaks. Strand breaks in the liver DNA of pair-fed controls or ethanol-fed rats were determined after 3 day and 1, 4 and 6 week periods of feeding as described in Materials and methods; each data point is the mean ± SE from four animals. *, Significantly different from the control value at 3 days at P < 0.05; ** and ***, significantly different from the corresponding control value at P < 0.05 or P < 0.01, respectively.

 
Inhibitory effects of antioxidant treatments
Free radical production.
The generation of 1-hydroxyethyl radicals following an acute dose of ethanol can be inhibited significantly by pre-treatment with vitamin C or vitamin E as shown in Figure 6Go.



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Fig. 6. The inhibitory effect of antioxidant pre-treatment (vitamin C or vitamin E) on the hepatic generation of 1-hydroxyethyl–POBN adducts after acute ethanol treatment. Groups of four rats (~ 200 g body wt) were given vitamin C (400 mg/kg, i.p., daily for 5 days), vitamin E (100 mg/kg, i.p., daily for 5 days) or a comparable volume of vehicle. After pre-treatment, a single oral dose of ethanol (5 g/kg, i.g.) was administered; water was given to the control group. Bile samples were collected and analysed as described in Figure 1Go; each data point is the mean ± SE from four animals. * and**, significantly different from the corresponding control value at P < 0.05 or P < 0.01, respectively.

 
Vitamin C pre-treatment (400 mg/kg, i.p., daily for 5 days) significantly reduced the signal intensity of ethanol-generated 1-hydroxyethyl radicals by ~28%. The inhibitory effect was more pronounced when vitamin E pre-treatment (100 mg/kg, i.p., daily for 5 days) was employed; this decreased the formation of 1-hydroxyethyl radicals by ~53%.

DNA strand breaks.
Pre-treatment with the antioxidants vitamins C or E as described above, also prevented the DNA strand breaks induced by acute ethanol treatment, reducing the levels to approximately those seen in the controls (Figure 7Go).



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Fig. 7. Effects of antioxidant pre-treatment (vitamin C or vitamin E) on strand breaks induced in hepatic DNA by acute ethanol treatment. Groups of five rats (200 g body wt) were given vitamin C (400 mg/kg, i.p., daily for 5 days), vitamin E (100 mg/kg, i.p., daily for 5 days) or the comparable volume of vehicle as detailed in Figure 6Go. Ethanol (5 g/kg, i.g.) was given to all groups except the controls, which were given water. The incidence of DNA strand breaks was determined 6 h after treatment as described in Materials and methods; each data point is the mean ± SE from four to six animals. * and **, significantly different from ethanol-treated value at P < 0.05 or from the control value at P < 0.05.

 
Discussion
The present study supports previous observations on the generation of 1-hydroxyethyl radicals by ethanol in vivo and further demonstrates that the production of 1-hydroxyethyl radicals can be prevented by antioxidants (vitamins C or E). It also shows that the generation of free radicals, possibly including 1-hydroxyethyl radicals and lipid radicals, can be detected in vivo after low levels of ethanol exposure as observed after 1 week of feeding with a liquid diet containing 5% (w/v) ethanol.

It has been reported that the metabolic activation of high concentrations of ethanol are mediated predominantly by the microsomal ethanol oxidizing system (MEOS), because this cytochrome P-450 has a high Km value for ethanol oxidation (39,40). ROS including O2.– and H2O2 can be generated in microsomes during NADPH-dependent electron transfer and the oxidation of the ethanol metabolite, acetaldehyde, via the catalytic activities of xanthine oxidase and aldehyde oxidase (26). These reactive agents may serve as precursors of OH. and, in the presence of ethanol, will eventually lead to the production of 1-hydroxyethyl radicals. Thus, it is possible that ethanol-generated ROS derived from MEOS and acetaldehyde play an important role in the formation of 1-hydroxyethyl radicals following acute ethanol exposure.

Ethanol is a strong inducer of CYP 2E1. This isozyme has a high NADPH oxidase activity leading to the production of large amounts of O2.– and H2O2 (41) and in agreement with this, ethanol-treated microsomes produced a highly significant generation of O2.– and OH. in an in vitro system, as shown using spin-trapping ESR techniques (1). CYP 2E1 may thus play a role in the generation of 1-hydroxyethyl radicals and this association was demonstrated in vitro (42). We have demonstrated in a time-course study of the 5% (w/v) ethanol liquid diet model using western immunobloting, that the maximum induction of the CYP 2E1 protein occurred at 1 week (P.Navasumrit, P.J.O'Connor, N.J.Nair, N.Frank and H.Bartsch, manuscript in preparation). This sequence of events was comparable with that seen for the induction of ethanol-generated free radicals as determined by the signal intensity of POBN adduct in bile, which also occurred at 1 week and together they indicate a possible correlation between the induction of CYP 2E1 and the in vivo generation of free radicals. In this study, POBN adduct signals were also observed in the bile of pair-fed controls. These were probably due to free radicals derived from endogenous lipid peroxidation products which also interact with POBN to produce POBN adducts and give an ESR spectrum fairly similar to that of the 1-hydroxyethyl–POBN adduct, as reported by Buettner (38). POBN adducts in pair-fed control animals have also been reported by others (19).

Data from this current study also indicate that ethanol induces DNA strand breaks in rat liver. An increased incidence of DNA strand breaks was observed both in the acute treatment model and also at a lower level of ethanol exposure when a liquid diet containing ethanol (5%; w/v) was administered. This was in agreement with previous reports in which ethanol-induced DNA breaks were observed when rat hepatocytes were incubated in cell culture (28) and in rat brain cells following a single oral dose of ethanol (27). The underlying mechanism of acute ethanol-induced hepatic DNA strand breaks may involve the effects of ethanol oxidation which contributes to the generation of ROS and acetaldehyde, as discussed above. The reasons for the increase in strand breaks in the control animals are less clear. They may arise partially as a result of maturation (i.e. age dependent) and possibly as an effect of the restricted caloric intake.

The effect of acetaldehyde in the production of DNA strand breaks may be mediated via the generation of free radicals by aldehyde oxidase as suggested by Fridovich (26); these reactive radicals can then attack DNA, leading to cleavage of the DNA backbone (43). Other possibilities for the production of acetaldehyde-induced DNA breaks may involve cytogenetic events following the formation of DNA–DNA or DNA–protein crosslinks (31,32). Acetaldehyde is also a highly reactive electrophile and can react with DNA to form DNA adducts, e.g. N2-ethyl-2'-deoxyguanosine which can be detected in granulocyte DNA from alcoholic patients and when human buccal epithelial cells are exposed to acetaldehyde (4446). The removal or repair of such DNA lesions may then initiate the production of DNA strand breaks.

A significant induction of hepatic DNA strand breaks was also observed in ethanol-treated rats throughout a 6 week period of maintenance on a liquid diet containing ethanol (5%; w/v) which lead to blood ethanol concentrations of 153 mg/dl (data not shown); this increased frequency of strand breakage may thus reflect the situation that occurs in chronic alcoholics. From a time-course study, the maximum incidence of ethanol-induced DNA strand cleavage was observed at 1 week and was correlated with the profile of ethanol-generated free radicals. The evidence, therefore, also supports a possible association between the generation of free radicals and the formation of hepatic DNA strand breaks. In agreement with these observations, others have also reported a significant correlation between the magnitude of ethanol-derived radicals, the 1-hydroxyethyl radical, and the severity of liver pathology (3,47).

It seems possible therefore, that these ethanol-induced hepatic DNA strand breaks could be mediated by free radical formation resulting from the induction of CYP 2E1. The potentially important role of CYP 2E1 in this sequence of events leading to the formation of DNA strand breaks was demonstrated by Kukielka and Cederbaum (8). They showed that incubation of plasmids with ethanol-treated microsomes containing NADPH or NADH and iron (in order to generate OH.), resulted in the formation of DNA strand breaks and that this effect was inhibited completely when anti-CYP 2E1 IgG was added into the reaction mixture.

The antioxidant pre-treatment data clearly indicated that both vitamin C or E can prevent the generation of 1-hydroxyethyl radicals following acute ethanol treatment. The mechanisms involved in these inhibitory effects against 1-hydroxyethyl radical formation, however, are not well established. These antioxidants probably exert their effects through their ability to scavenge reactive oxidants. Vitamin C is a hydrophilic antioxidant and also a strong reducing agent which reacts rapidly with O2.– and more rapidly with OH. (48). Consequently, vitamin C pre-treatment could prevent 1-hydroxyethyl radical formation by quenching reactive oxyradical intermediates. In addition, Stoyanovsky et al. (49) reported that vitamin C inhibited the formation of the 1-hydroxyethyl/PBN adduct and rapidly reduced the pre-formed adduct in an in vitro system. Vitamin E also reacts with oxygen radicals, i.e. with the superoxide-generating system; however, the efficiency of radical scavenging by vitamin E depends on the attacking radical species. It has been shown that vitamin E is a potent scavenger of peroxyl radicals, but it is unlikely that it scavenges hydroxyl radicals efficiently in vivo (16). In the present study, vitamin E pre-treatment (100 mg/kg; i.p., 5 days) significantly inhibited ethanol-generated 1-hydroxyethyl radicals and the inhibitory effect was more pronounced compared with vitamin C pre-treatment (400 mg/kg., i.p., 5 days): it is possible, therefore, that 1-hydroxyethyl radicals are generated within the vicinity of a lipid compartment of the cell. In agreement with the observation that CYP (which is located at the membrane of the smooth endoplasmic reticulum) is the active site for the generation of reactive oxygen intermediates, these studies showed a correlation between the induction of CYP 2E1 and the generation of 1-hydroxyethyl radicals. Furthermore, a recent observation using a laser scanner confocal fluorescence image analysis system demonstrated that 1-hydroxyethyl radical adducts were present on the plasma membrane of acute ethanol-treated hepatocytes (50). Accordingly, the potent lipophilic antioxidant, vitamin E showed a greater inhibitory effect on acute ethanol-generated 1-hydroxyethyl radicals, compared with the hydrophilic antioxidant, vitamin C. Although vitamin E is not a strong scavenger of the reactive oxygen radical intermediates of 1-hydroxyethyl radical formation (i.e. OH.), it may act via a direct scavenging of 1-hydroxyethyl radicals.

Present data also demonstrate that ethanol-induced hepatic DNA strand break production was completely inhibited by antioxidants. Ethanol, therefore, may induce DNA strand breaks through the generation of reactive oxidants since the prevention of ethanol-induced hepatic DNA strand breaks by pre-treatment with vitamins C or E was correlated with the inhibitory effects of these pre-treatments on the generation of 1-hydroxyethyl radicals after acute ethanol exposure. Although a direct effect of 1-hydroxyethyl radicals in the generation of DNA damage is still not well established, the results obtained in this study imply an association between the generation of 1-hydroxyethyl radicals and the appearance of an increased incidence of hepatic DNA strand breaks.


    Acknowledgments
 
This work was supported by the Cancer Research Campaign (CRC), UK and by a grant to P.N. from the government of Thailand.


    Notes
 
3 Present address: Chulabhorn Research Institute, Bangkok 10210, Thailand Back

5 To whom correspondence should be addressed Email: pjoconnor{at}picr.man.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 References
 

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Received March 31, 1999; revised September 1, 1999; accepted September 29, 1999.