Synergistic Interaction between Hydroquinone and ... fileSeveral reports on the detoxification of...
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[CANCER RESEARCH 45, 4853-4857, October 1985] Synergistic Interaction between Hydroquinone and Acetaldehyde in the Induction of Sister Chromatid Exchange in Human Lymphocytes in Vitro* Susan Knadle2 Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, San Francisco, CA 94143 ABSTRACT Hydroquinone, a metabolite of benzene, and acetaldehyde, a metabolite of ethanol, induce sister chromatid exchanges (SCEs) at relatively high concentrations. Because both compounds are reported to form glutathione conjugates, experiments were car ried out to see if there was a synergistic effect when lower concentrations of both chemicals were added to human lympho cyte cultures. Hydroquinone (40 Õ¿M) increased SCEs in some individuals but not in others. However, the rate of SCE was more than doubled when cells were pretreated with diethyl maléate, which transiently depletes cellular glutathione. Acetaldehyde by itself increased SCEs at 100 ¿tM, but it increased SCEs at 1 /¿M inthe presence of diethyl maléate.When various concentrations of acetaldehyde were added to cultures containing 40 fiM hydro- quinone, synergism in the induction of SCEs was observed. The lowest effective concentration of acetaldehyde varied among individuals from 1 to 100 /«M. These observations suggest that glutathione is involved in the detoxification of hydroquinone and acetaldehyde in lymphocytes and that the simultaneous pres ence of both chemicals may saturate this mechanism and thus increase their genotoxic potency. Genetic differences in glutathi one metabolism may govern the concentration of acetaldehyde at which synergism occurs in different individuals. INTRODUCTION Hydroquinone, a metabolite of benzene, and acetaldehyde, a metabolite of ethanol, can be found in the blood after in vivo exposure to the parent compounds (1, 2). Benzene is metabo lized by the cytochrome P-450 enzymes of the liver; the final metabolites consist primarily of glucuronic acid, sulfate, and mercapturic acid conjugates (3-5). Free phenol, catechol, and hydroquinone have also been found in blood and bone marrow after inhalation of benzene (2). Furthermore, hydroquinone and catechol persisted in blood and bone marrow for at least 9 h after exposure stopped (2). Ethanol is also metabolized in the liver. Alcohol dehydrogenase enzymes are primarily responsible for the metabolism of ethanol, although cytochrome P-450 en zymes also oxidize ethanol during chronic ingestion (6). The concentrations of its metabolite, acetaldehyde, have been mea sured in human blood after ethanol administration (1). The max imum concentrations were 43 MM in alcoholics and 27 UM in control subjects during a period when the concentration of ethanol in the blood decreased from about 50 to 25 mw. Exposure to benzene produces leukopenia in peripheral blood, 1This work was supported by the U. S. Department of Energy (DE-AC03-76- SF01012) and by National Institutes of Health National Service Award 5 T32 ES07106 from the National Institute of Environmental Health Sciences. 2 Current address: Environmental Health Associates, Inc., 520 Third Street, Suite 208, Oakland, CA 94607. Received 2/11/85; revised 7/2/85; accepted 7/5/85. bone marrow, and spleen. Although the ingestion of ethanol by itself has no effect on leukocyte counts, its ingestion during benzene exposure potentiates the leukopenia produced by ben zene inhalation (7). These observations have been attributed to induction of similar forms of cytochrome P-450 by ethanol and benzene. Another explanation, which is explored in this study, is that ethanol or its metabolites can interact synergistically with the myelotoxic metabolites of benzene to increase leukotoxicity. Several reports on the detoxification of these compounds (8- 10) suggest that glutathione may mediate this potentiation of leukotoxicity. The metabolism of benzene or phenol by hepatic microsomes produces adducts that are covalently bound to microsomal protein, and a covalently bound species arises from the formation of hydroquinone or its oxidation product, p-ben- zoquinone (10). Addition of glutathione to the incubation mixture reduces the binding by 90-95% and produces a glutathione conjugate of hydroquinone (10, 11). Acute alcohol intoxication results in a decrease in glutathione concentration (9), as well as an increase in lipid peroxidation (12). Acetaldehyde may mediate this form of toxicity, because 50 to 100 ^M acetaldehyde admin istered to isolated hepatocytes decreased the glutathione con tent of the cells in a dose-dependent manner from 72 to 57% of control values (13). Furthermore, glutathione is capable of spon taneously reacting with acetaldehyde in a cell-free system (13). Both hydroquinone (14) and acetaldehyde (15) have been reported to induce SCE3 in human lymphocytes. Therefore, this study was undertaken to determine if a synergistic interaction might occur in lymphocytes exposed to these compounds si multaneously. Since glutathione is reported to detoxify both of these compounds, its role in increasing the SCE rate caused by hydroquinone and acetaldehyde, individually and together, was investigated. Pretreatment with diethyl maléatewas used to transiently reduce cellular glutathione (16) and examine the role of glutathione in preventing DNA damage by these chemicals. MATERIALS AND METHODS Chemicals. Lymphocytes in whole blood were cultured in the pres ence of one of the following: acetaldehyde (Aldrich Chemical Co., Mil waukee, Wl) at concentrations of from 1 to 100 MM;hydroquinone (Sigma Chemical Co., St. Louis, MO) at 40 A/M; acetaldehyde plus 40 MM hydroquinone; diethyl maléate(Sigma Chemical Co.) at 50 (<M; 50 IM diethyl maléateplus acetaldehyde; or 50 /IM diethyl maléateplus 40 MM hydroquinone. In all cases, appropriate solvent controls were tested concurrently. Diethyl maléatewas first dissolved in 100% ethanol, and 10 n\ were then added to each culture. The final solvent concentration was 2 MM. Acetaldehyde and hydroquinone were freshly prepared in ice-cold Ros- well Park Memorial Institute (RPMI) Tissue Culture Medium 1640 without serum. Acetaldehyde dilutions were prepared in Reacti-vials (Pierce Chemical Co., Rockford, IL) sealed with Tuf-Bond Teflon-silicone bonded 3 The abbreviation used is: SCE, sister chromatid exchange. 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[CANCER RESEARCH 45, 4853-4857, October 1985]
Synergistic Interaction between Hydroquinone and Acetaldehyde in theInduction of Sister Chromatid Exchange in Human Lymphocytes in Vitro*
Susan Knadle2
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, San Francisco, CA 94143
ABSTRACT
Hydroquinone, a metabolite of benzene, and acetaldehyde, ametabolite of ethanol, induce sister chromatid exchanges (SCEs)at relatively high concentrations. Because both compounds arereported to form glutathione conjugates, experiments were carried out to see if there was a synergistic effect when lowerconcentrations of both chemicals were added to human lymphocyte cultures. Hydroquinone (40 Õ¿M)increased SCEs in someindividuals but not in others. However, the rate of SCE was morethan doubled when cells were pretreated with diethyl maléate,which transiently depletes cellular glutathione. Acetaldehyde byitself increased SCEs at 100 ¿tM,but it increased SCEs at 1 /¿Min the presence of diethyl maléate.When various concentrationsof acetaldehyde were added to cultures containing 40 fiM hydro-
quinone, synergism in the induction of SCEs was observed. Thelowest effective concentration of acetaldehyde varied amongindividuals from 1 to 100 /«M.These observations suggest thatglutathione is involved in the detoxification of hydroquinone andacetaldehyde in lymphocytes and that the simultaneous presence of both chemicals may saturate this mechanism and thusincrease their genotoxic potency. Genetic differences in glutathione metabolism may govern the concentration of acetaldehydeat which synergism occurs in different individuals.
INTRODUCTION
Hydroquinone, a metabolite of benzene, and acetaldehyde, ametabolite of ethanol, can be found in the blood after in vivoexposure to the parent compounds (1, 2). Benzene is metabolized by the cytochrome P-450 enzymes of the liver; the final
metabolites consist primarily of glucuronic acid, sulfate, andmercapturic acid conjugates (3-5). Free phenol, catechol, and
hydroquinone have also been found in blood and bone marrowafter inhalation of benzene (2). Furthermore, hydroquinone andcatechol persisted in blood and bone marrow for at least 9 hafter exposure stopped (2). Ethanol is also metabolized in theliver. Alcohol dehydrogenase enzymes are primarily responsiblefor the metabolism of ethanol, although cytochrome P-450 en
zymes also oxidize ethanol during chronic ingestion (6). Theconcentrations of its metabolite, acetaldehyde, have been measured in human blood after ethanol administration (1). The maximum concentrations were 43 MM in alcoholics and 27 UM incontrol subjects during a period when the concentration ofethanol in the blood decreased from about 50 to 25 mw.
Exposure to benzene produces leukopenia in peripheral blood,
1This work was supported by the U. S. Department of Energy (DE-AC03-76-
SF01012) and by National Institutes of Health National Service Award 5 T32ES07106 from the National Institute of Environmental Health Sciences.
2Current address: Environmental Health Associates, Inc., 520 Third Street,
Suite 208, Oakland, CA 94607.Received 2/11/85; revised 7/2/85; accepted 7/5/85.
bone marrow, and spleen. Although the ingestion of ethanol byitself has no effect on leukocyte counts, its ingestion duringbenzene exposure potentiates the leukopenia produced by benzene inhalation (7). These observations have been attributed toinduction of similar forms of cytochrome P-450 by ethanol and
benzene. Another explanation, which is explored in this study, isthat ethanol or its metabolites can interact synergistically withthe myelotoxic metabolites of benzene to increase leukotoxicity.
Several reports on the detoxification of these compounds (8-
10) suggest that glutathione may mediate this potentiation ofleukotoxicity. The metabolism of benzene or phenol by hepaticmicrosomes produces adducts that are covalently bound tomicrosomal protein, and a covalently bound species arises fromthe formation of hydroquinone or its oxidation product, p-ben-
zoquinone (10). Addition of glutathione to the incubation mixturereduces the binding by 90-95% and produces a glutathione
conjugate of hydroquinone (10, 11). Acute alcohol intoxicationresults in a decrease in glutathione concentration (9), as well asan increase in lipid peroxidation (12). Acetaldehyde may mediatethis form of toxicity, because 50 to 100 ^M acetaldehyde administered to isolated hepatocytes decreased the glutathione content of the cells in a dose-dependent manner from 72 to 57% of
control values (13). Furthermore, glutathione is capable of spontaneously reacting with acetaldehyde in a cell-free system (13).
Both hydroquinone (14) and acetaldehyde (15) have beenreported to induce SCE3 in human lymphocytes. Therefore, this
study was undertaken to determine if a synergistic interactionmight occur in lymphocytes exposed to these compounds simultaneously. Since glutathione is reported to detoxify both ofthese compounds, its role in increasing the SCE rate caused byhydroquinone and acetaldehyde, individually and together, wasinvestigated. Pretreatment with diethyl maléatewas used totransiently reduce cellular glutathione (16) and examine the roleof glutathione in preventing DNA damage by these chemicals.
MATERIALS AND METHODS
Chemicals. Lymphocytes in whole blood were cultured in the presence of one of the following: acetaldehyde (Aldrich Chemical Co., Milwaukee, Wl) at concentrations of from 1 to 100 MM;hydroquinone (SigmaChemical Co., St. Louis, MO) at 40 A/M; acetaldehyde plus 40 MMhydroquinone; diethyl maléate(Sigma Chemical Co.) at 50 (<M; 50 IMdiethyl maléateplus acetaldehyde; or 50 /IM diethyl maléateplus 40 MMhydroquinone. In all cases, appropriate solvent controls were testedconcurrently.
Diethyl maléatewas first dissolved in 100% ethanol, and 10 n\ werethen added to each culture. The final solvent concentration was 2 MM.Acetaldehyde and hydroquinone were freshly prepared in ice-cold Ros-
well Park Memorial Institute (RPMI) Tissue Culture Medium 1640 withoutserum. Acetaldehyde dilutions were prepared in Reacti-vials (PierceChemical Co., Rockford, IL) sealed with Tuf-Bond Teflon-silicone bonded
3The abbreviation used is: SCE, sister chromatid exchange.
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SCEs INDUCED BY HYDROQUINONE PLUS ACETALDEHYDE
septa (Pierce Chemical Co.). Acetaldehyde was transferred by a Hamiltonsyringe to culture bottles sealed with Teflon-silicone septa.
Culture Techniques. Whole blood from seven healthy individuals wasadded to RPMI 1640 containing 10% fetal bovine serum (Grand IslandBiological Co., Grand Island, NY), 1% phytohemagglutinin (PHA-M; Difco
Laboratories, Detroit, Ml), penicillin (100 uniis/ml), streptomycin (100 ¡tglml), 2 niM glutamine (Grand Island Biological Co.), and 20 pM 5-bromo-
deoxyuridine. The blood was diluted 1:25 with RPM11640 and dispensedinto 1-ounce culture bottles so that each culture contained 0.2 ml blood
and 4.8 ml culture medium. Acetaldehyde, hydroquinone, and diethylmaléatewere added at 17 h of culture. Diethyl maléatewas alwaysadded 15 min before acetaldehyde or hydroquinone, and hydroquinonewas added 15 min before acetaldehyde.
Cultures were incubated in complete darkness for 72 h at 37°C.Two
h before fixation, Cdcemid at a final concentration of 0.2 IM was addedto each culture. The cells were centrifugea at 800 x g for 5 min, and themedium was aspirated. Cells were resuspended in a hypotonie solution,0.075 M KCI, for 20 min to lyse the erythrocytes and spread thechromosomes. They were again centrifuged and fixed in rnethanol: aceticacid (3:1). After two changes of fixative, the cells were suspended in asmall amount of fixative and dropped onto warm, wet microslides, whichwere dried rapidly on a 55°Cslide warmer.
The slides were stained for 20 min in a solution of Hoechst 33258(0.05 ng/ml) in 0.15 M Sorensen's buffer. pH 6.8. The slides were washed,
dried, and covered with buffer, and a coverslip was floated on top. Theywere then exposed to 360-nm wavelength light at a distance of 2 cm
(approximately 20 J/rrf/s) for 6 to 12 min, stained in 2% Giernsa inSorensen's buffer, air-dried, and permanently mounted. SCEs wereanalyzed in 50 second-division metaphases for each point. Student's f-
test was used for statistical analysis. All of the studies with acetaldehydeplus hydroquinone were replicated in the same individual in two separate
experiments.
RESULTS
Effect of Acetaldehyde and Hydroquinone on SCE Induction. At concentrations from 1 to 20 UM, acetaldehyde did notcause an increase in SCEs in human lymphocytes (Table 1).However, at 100 MM acetaldehyde, a significant increase (P <0.05) in SCEs was observed in cells from each of the individualstested.
Hydroquinone at a concentration of 40 MM induced SCEs incells from some individuals (P < 0.05) but not from others (Table2). The qualitative response of the lymphocytes from specificindividuals was reproducible upon retesting, although the exactnumber of induced SCEs varied somewhat.
Table 1
Induction of SCEs by acetaldehyde
0.2 ml whole blood was cultured in 4.8 ml RPM11640 medium containing 10%fetal bovine serum, 1% PHA-M, penicillin(100 units/ml), streptomycin (100 ^g/ml),2 mMglutamine, and 20 UM5-bromodeoxyuridine.Acetaldehydewas added at 17h of culture, and Coteemid,at a final concentration of 0.2 nu, was added 2 h beforeharvest at 72 h.
Table 2Induction of SCEs by hydroquinone
Cells were cultured as described in Table 1. Hydroquinone was added at 17 hof culture.
Acetaldehyde(MM)0121020100DonorA6.7
±0.3"7.0
±0.37.0±0.37.1±0.47.9+0.416.0±0.7CSCEs/cell'Donor
B9.5
±0.58.9±0.49.1±0.410.0±0.69.7+0.514.7±0.7CDonor
C8.4
±0.48.5±0.48.4±0.48.7±0.48.3±0.416.5±0.8°
SCEs/cell"DonorAB
CDE
FUntreated8.5
±0.4"
9.0 ±0.57.3 ±0.49.6 ±0.59.5 ±0.48.6 ±0.4Hydroquinone(40
MM)11.2±0.4C
8.8 ±0.47.3 ±0.4
12.0±0.5C12.6±0.6C
7.9 ±0.4
" Fifty cells/point." Mean ±SE.c Significantlydifferent from control (P < 0.05, paired Student's Mest).
* Fifty cells/point.6 Mean ±SE.c Significantlydifferent from control (P < 0.05).
When acetaldehyde, at concentrations that alone did not induce SCEs, was added to lymphocyte cultures containing 40 MMhydroquinone, an increase in SCEs above control frequencieswas observed in the cells from all individuals tested (Tables 3-
5). Furthermore, the observed number of SCEs was greater thanexpected for an additive effect. The expected number of SCEswas the sum of the number of SCEs above control found aftertreatment with hydroquinone alone and the number observedafter acetaldehyde alone at each concentration. In lymphocytesfrom Donor B (Table 3), acetaldehyde alone at 2,10, and 20 U.Mdid not induce SCEs above control values. However, acetaldehyde at these concentrations plus hydroquinone increased thenumber of SCEs per cell above additive levels in a dose-depend
ent manner in both experiments. In lymphocytes from Donor C(Table 4), however, 2 MM acetaldehyde plus hydroquinone didnot increase SCEs above control values. However, when theacetaldehyde concentration was 10 MMand hydroquinone wasalso present, there was an increase of about 5 SCEs per cell inboth experiments. At the highest concentration of acetaldehyde,100 MM, when hydroquinone was present, the SCE frequencywas about doubled in both experiments. In the lymphocytes fromDonor G (Table 5), significant synergism did not occur untilacetaldehyde alone, at 100 MM,induced an increase in the SCEfrequency. At this concentration, with hydroquinone, the SCEfrequency almost doubled.
Thus, the synergism between acetaldehyde and 40 MMhydroquinone occurred at different acetaldehyde concentrations in thelymphocytes from the three donors (Tables 3-5). Synergism
occurred at 1 MM acetaldehyde in the lymphocytes from oneindividual, at 10 MM in another, and at 100 MM in the third. Asimilar variation in the concentration of acetaldehyde at whichsynergism occurred was observed in the lymphocytes of threeother individuals tested (data not shown).
Effect of Diethyl MaléatePretreatment on SCE Inductionby Hydroquinone. To assay the effect of a depletion of glutathi-
one on the number of SCEs induced by hydroquinone, lymphocyte cultures were treated with 50 MM diethyl maléate15 minbefore the addition of 40 MMhydroquinone. As shown in Table6, the number of SCEs induced by hydroquinone was consistently more than doubled when glutathione was transiently depleted. In two individuals (Donors A and B), diethyl maléatealoneled to an increase in SCEs (P < 0.05), and in one individual(Donor A), hydroquinone alone also induced an increase in SCEs.The magnitude of these increases, however, was small compared to the increase due to diethyl maléatepretreatment plus
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TablesInduction of SCEsby acetaldehydeplus hydroquinone(DonorB)
Cells were cultured as described in Table 1. Hydroquinone (HQ) was added at 17 h of culture, and acetaldehyde(CH3CHO)was added 15 min later.
Experiment1TreatmentNone40
MMHQ1MMCHsCHO1MMCHsCHO +HQ2MMCHsCHO2MMCHsCHO +HQ10
MMCHaCHO10MMCHaCHO +HQ20MMCHaCHO20MMCHaCHO +HQ100
MMCHaCHO100MMCHaCHO + HQObservedSCEs/ceH"9.0
±0.5"8.8
±0.49.0±0.411.
3±0.6°8.8
±0.413.0±0.6C9.2
±0.616.2±0.7C9.3
±0.616.6±0.7C14.4
±0.5e25.0±1.0eExpectedSCEs/cell9.08.89.29.314.4Relativeincrease2.34.27.07.310.6Experiment
2ObservedSCEs/cell9.5
±0.510.6±0.78.9±0.412.2±0.5°9.1
±0.513.4±0.5C10.0
±0.516.1±0.7°9.7
±0.4a1
4.3 ±0.7egExpectedSCEs/cell10.010.211.1Relativeincrease2.23.25.0
" Fifty cells/point.6 Mean ±SE.c Significantlydifferent from acetaldehydeatoneor hydroquinonealone (P < 0.05)." Too few second-divisioncells to score.8 Significantlydifferent from control (P < 0.05).
Table4Induction of SCEsby acetaldehydeplus hydroquinone(DonorC)
Cells were cultured as described in Table 1. Hydroquinone (HQ) was added at 17 h of culture, and acetaldehyde(CHaCHO)was added 15 min later.
Experiment1TreatmentNone
40 MMHQ2 MMCHaCHO2 MMCHsCHO + HQ10 MMCHsCHO10 MMCHsCHO +HQ20 MMCHsCHO20 MMCHsCHO + HQ100 MMCHsCHO100 MMCHaCHO + HQObserved
SCEs/cell"8.4
±0.3*8.5
±0.4c8.5
±0.48.2 ±0.4
13.6 ±0.6"
8.4 ±0.414.3 ±0.55a10.3 ±0.6e23.5 ±0.6aExpected
SCEs/cell8.5
8.3
8.510.4Relative
increase0
5.3
5.813.1Experiment
2Observed
SCEs/cell8.4
±0.48.3 ±0.48.4
±0.48.7 ±0.4
14.0 ±0.5a
8.3 ±0.415.7 ±0.5d16.5 ±0.8e25.0 ±1.0"Expected
SCEs/cell8.4
8.7
8.3
16.5Relative
increase0
5.3
7.48.5
* Fifty cells/point.6 Mean ±SE.c Not scored." Significantlydifferent from acetaldehydeatoneor hydroquinonealone (P < 0.05)." Significantlydifferent from control (P < 0.05).
TablesInduction of SCEsby acetaldehydeplus hydroquinone(Donor G)
Cells were cultured as described in Table 1. Hydroquinone (HQ) was added at 17 h of culture, and acetaldehyde(CHaCHO)was added 15 min later.
Experiment1TreatmentNone40
MMHQ10 MMCHsCHO10 MMCHaCHO + HQ20 MMCHaCHO20 MMCHaCHO + HQ100 MMCHsCHO100 MMCHsCHO -t-HQObserved
SCEs/cell88.1
±0.5611.3
±0.4c8.4
±0.48.2 ±0.4
10.4 ±0.6"15.0 ±0.8"27.4 ±0.8'Expected
SCEs/cell11.4
18.2Experiment
2Relative
Observedincrease SCEs/cell8.1
±0.49.6 ±0.58.9
±0.48.5 ±0.4
10.3 ±0.5"17.9 ±0.8e
9.2 26.2 ±0.7'ExpectedSCEs/cell10.019.4Relative
increase0.3
6.8* Fifty cells/point.* Mean ±SE.c Not scored.
Significantlydifferent from acetaldehydealone (P < 0.01).* Significantlydifferent from control (P < 0.05).' Significantlydifferent from acetaldehydealone or hydroquinoneatone(P < 0.05).
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Table 6Induction of SCEsby hydroquinoneafter pretreatment with diethyl maléate
Cellswere cultured as described in Table 1. Diethyl maléatewas added at 17 hof culture, and hydroquinonewas added 15 min later.
SCEs/cell"
TreatmentNone
Hydroquinone(40 MM)Diethyl maléate(50 MM)Diethyl maléate(50 MM)t
hydroquinone(40 MM)Donor
A8.5±0.46
11.2±0.4C13.0±0.5C28.6 ±0.8°Donor
B9.0
±0.58.8 ±0.4
12.9 ±0.6°43.7 ±1,04CDonor
F8.6
±0.47.9 ±0.48.5 ±0.5
19.4 ±0.5°
' Fifty cells/point.6 Mean ±SE.c Significantlydifferent from control (P < 0.05).
Table 7Induction ot SCEsby acetaldehydeafter pretreatment with diethyl maléate
Cells were cultured as described in Table 1. Diethyl maléate(DEM)was addedat 17 h of culture, and acetaldehyde(CH3CHO)was added 15 min later.
SCEs/cell"
Treatment Donor A Donor B
None50MMDEM1
MMCH3CHO1MMCH3CHO+DEM2MMCH3CHO2MMCH3CHO+DEM10MMCH3CHO10MMCH3CHO
+DEM20MMCH3CHO20MMCH3CHO+DEM100iiMCH3CHO100MMCH3CHO
+ DEM6.7
±0.3"9.7±0.4C7.0
±0.313.2±0.7C"7.0
±0.313.0±0.5c-d7.1
±0.413.4±0.4c-d7.9
±0.417.9±0.6c-<(16.0±0.7C38.0±1.0c'd9.5
±0.510.7±0.6C8.9
±0.412.1±0.5C-"9.1
±0.412.0±0.5c'd10.0
±0.613.9 ±0.6e"9.7
±0.515.2±0.1C'C(14.7
±0.7C26.2±0.8c-d
* Fifty cells/point.6 Mean ±SE.c Significantlydifferent from control (P < 0.05)." Significantlydifferent from diethyl maléatealone (P < 0.05).
hydroquinone.Effect of Diethyl MaléatePretreatment on SCE Induction
by Acetaldehyde. Lymphocyte cultures were treated with 50^M diethyl maléate15 min before the addition of various concentrations of acetaldehyde. Acetaldehyde alone did not cause anincrease in SCEs above control values until the concentrationreached 100 UM, but treatment with diethyl maléatebefore 1 UMacetaldehyde induced an increase in SCEs (Table 7). The numbers of SCEs induced after pretreatment with diethyl maléateincreased in a dose-dependent manner with increasing concen
trations of acetaldehyde in both individuals. Although 100 A/Macetaldehyde alone induced SCEs above control values, depletion of glutathione by pretreatment with diethyl maléatestillproduced a synergistic effect at the concentration; the numberof SCEs in one individual (Donor A) was more than doubled, andthe number in the other (Donor B) was almost doubled.
DISCUSSION
The results of this study indicate that acetaldehyde and hydroquinone interact synergistically to increase the frequency ofSCEs in human lymphocytes in vitro. The induction of SCEs is asensitive indicator of the production of lesions in DMA (17) andcan be considered an indicator of a compound's ability to cause
genotoxicity in lymphocytes. Since hydroquinone and acetaldehyde can be found in blood after exposure to benzene andethanol, the synergistic increase in SCE frequency in lympho
cytes in vitro suggests that simultaneous in vivo exposure tobenzene and ethanol could be more genotoxic than exposure toeither chemical separately. Furthermore, ethanol and benzenehave been demonstrated to induce identical forms of cytochromeP-450 (18), and ethanol increases the blood clearance rates of
benzene owing to the induction of benzene metabolism (19).Therefore, the leukemogenic potential of benzene may be increased by ingestion of ethanol.
The mechanism underlying this synergistic interaction appearsto involve glutathione, because the induction of SCEs by eitheracetaldehyde or hydroquinone was potentiated when glutathionewas depleted by diethyl maléate(Tables 6 and 7). Since aglutathione conjugate of hydroquinone or its oxidized form, p-benzoquinone, has been identified (11), the presence of glutathione in lymphocytes may be expected to competitively inhibitthe binding of hydroquinone to cellular macromolecules. Conversely, the depletion of glutathione would allow hydroquinoneto interact with DNA to produce lesions that lead to SCEs.
It has been proposed that acetaldehyde forms a thiohemiacetalwith glutathione, which provides a nontoxic reservoir of acetaldehyde that may be released later (20). The observation thatsulfhydryl compounds protect mice against acute alcoholic liverinjury (8, 9) led to the finding that acetaldehyde spontaneouslyreacts with glutathione in a cell-free system (13). The results of
these studies further suggested that glutathione may function toprotect enzymes, such as aldehyde dehydrogenase, from inac-tivation by reactive compounds, such as acetaldehyde itself.Formaldehyde and acetaldehyde (1-5 mw) have been shown toinactivate horse liver alcohol and aldehyde dehydrogenases in acell-free system (21). Therefore, depletion of glutathione by its
reaction with diethyl maléate(16) or hydroquinone would allowacetaldehyde to inactivate aldehyde dehydrogenase and therebyreduce its own detoxification.
This study also demonstrated that the synergistic interactionbetween acetaldehyde and hydroquinone occurs at various concentrations of acetaldehyde in the lymphocytes of different individuals. The lowest effective concentration of acetaldehyde atwhich this synergism occurred varied by a factor of 100 amongthe individuals tested. In the most sensitive lymphocytes (Table3), the lowest effective acetaldehyde concentration was 1 UM, aconcentration that is likely to be found frequently in human blood(1). In lymphocytes of intermediate sensitivity (Table 4), thelowest effective concentration of acetaldehyde was 10 /¿M;thisconcentration has also been reported to be reached in humanblood after administration of ethanol (1). In the least sensitivelymphocytes (Table 5), the lowest effective concentration ofacetaldehyde, 100 ^M, was not found in human blood even afterintravenous infusion of ethanol to peak blood concentrations of43-54 rriM (1). The lowest effective acetaldehyde concentration
at which synergism occurred in a particular individual was reproducible upon retesting, indicating that the variation in responseamong lymphocytes of individuals may have a genetic basis.Moreover, the increased induction of SCEs by either acetaldehyde or hydroquinone when glutathione was depleted suggeststhat such a possible genetic basis might reside in enzymes ofglutathione metabolism.
ACKNOWLEDGMENTS
The author wishes to thank SheldonWolff for his advice on the conduct of thisresearch and for critical reading of this manuscript. The excellent editorial assist-
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SCEs INDUCED BY HYDROQUINONE PLUS ACETALDEHYDE
ance of Mary McKenneyand the secretarial assistanceof Susan Brekhus are alsogreatly appreciated.
REFERENCES
1. Korsten, M. A., Matsuzaki, S., Feinman, L, and Lieber, C. High Woodacetal-dehyde levels after ethanol administration. N. Engl. J. Med., 292; 386-389,1975.
2. Rickert, D. E., Baker, T. S., Bus, J. S., Barrow, C. S., and Irons, R. D. Benzenedisposition in the rat after exposure by inhalation.Toxico!. Appi. Pharmacol.,49:417-423,1979.
3. Parke, D. V. and Williams, R. T. Studies in detoxification. 49. The metabolismof benzenecontaining [14C,]benzene.Brachem.J., 54: 231-238,1953.
4. Parke, D. V. and Williams, R. T. Studies in detoxification. 54. The metabolismof benzene, (a) The formation of phenylglutathione and phenylsulphuricacidfrom [14C]benzene.(i>)The metabolism of [14C]phenol.Bkxhem. J., 55: 337-
340,1953.5. Timbrel!,J. A. and Mitchell, J. R. Toxicity-related changes in benzene metab
olism in vivo. Xenobiotica, 7: 415-423,1977.6. Misra, P. S., Lefevre, A., Ishii, H., Rubin, E., and Lieber, C. S. Increase of
ethanol, meprobamate, and pentobarbital metabolism after chronic ethanoladministration in man and in rats. Am. J. Med., 57: 346-351,1971.
7. Baarson, K. A., Snyder, C. A., Green, J. D., Sellakumar,A., Goldstein, B. D.,and Albert, R. A. The hematotoxic effects of inhaled benzene on peripheralblood, bone marrow, and spleen cells are increased by ingested ethanol.Toxicol. Appi. Pharmacol.,64:393-404,1982.
8. Guerri, C., Godfrey, W., and Grisoha. S. Protection against toxic effects offormaldehydeIn vitro, and of methanolor formaldehydeIn vivo, by subsequentadministration of SH reagents. Physiol.Chem. Phys., fl: 543-550,1976.
9. MacDonald, C. M., Dow, J., and Moore, M. R. A possible protective role forsulfhydral compounds in acute alcoholic liver injury. Btochem.Pharmacol.,26;1529-1531,1977.
10. Tunek, A., Platt, K. L., Bentley, P., and Oesch, F. Microsomal metabolism of
benzene to species irreversibly binding to microsomal protein and effects ofmodifications of this metabolism. Mol. Pharmacol., 74: 920-929,1978.
11. Tunek, A., Platt, K. L., Przybylski, M., and Oesch, F. Multi-step metabolicactivation of benzene. Effect of Superoxidedismutase on covalent binding tomicrosomalmacromolecules,and identificationof glutathioneconjugates usinghigh pressure liquid chromatography and field desorption mass spectrometry.Chem.-Biol. Interact., 33:1-17,1980.
12. MacDonald,C. M. The effects of ethanol on hepatic lipid peroxidation and onthe activities of glutathione reducÃaseand peroxidase. FEBS Lett., 35: 227-230,1973.
13. Vina, J., Estrela, J. M., Guerri, C., and Romero, F. J. Effect of ethanol onglutathioneconcentration in isolated hepatocytes. Brachem.J., 788;549-552,1980.
14. Morimoto, K. and Wolff, S. Increase of sister chromatid exchanges andperturbations of cell division kinetics in human lymphocytes by benzenemetabolites. Cancer Res., 40:1189-1193,1980.
15. Jansson, T. The frequency of sister chromatid exchanges in human lymphocytes treated with ethanol and acetaldehyde.Hereditas,97: 301-303,1982.
16. Chasseaud, L. F. The nature and distribution of enzymes catalyzing theconjugationof glutathionewith foreigncompounds. Drug Metab. Rev.,2:185-220,1973.
17. Wolff, S. Sister chromatid exchangeas a test for mutageniccarcinogens.Ann.NY Acad. Sci., 407:142-153,1983.
18. Ingelman-Sundberg,M. and Hagbjork, A. L. On the significance of the cyto-chrome P-450-dependenthydroxyl radical-mediatedoxygénationmechanism.Xenobiotica, 72:673-686,1982.
19. Driscoll,K. E. and Snyder, C. A. The effects of ethanol ingestion and repeatedbenzeneexposures on benzenepharmacokinetics.Toxicol. Appi. Pharmacol.,73: 525-532,1984.
20. Ketterer, B. The role of nonenzymatic reactions of glutathione in xenobioticmetabolism. Drug Metab. Rev., 73:161-187,1982.
21. Guerri, C. and Grisolla,S. Influenceof prolonged ethanol intake on the levelsand turnover of alcoholand aldehyde dehydrogenasesand glutathione. In: H.Begleiter (ed.), BiologicalEffects of Alcohol, pp. 365-384. New York: PlenumPublishingCorp., 1978.
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in VitroHuman Lymphocytes Acetaldehyde in the Induction of Sister Chromatid Exchange in Synergistic Interaction between Hydroquinone and
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