Induction of class 3 aldehyde dehydrogenase in the mouse hepatoma cell line HEPA-1 by various chemicals

Chem -Bwl Interactwns, 83 (1992) 107-119

107

Elsewer Scientific Pubhshers Ireland Ltd

INDUCTION OF CLASS 3 ALDEHYDE DEHYDROGENASE IN THE MOUSE HEPATOMA CELL LINE HEPA-1 BY VARIOUS CHEMICALS RIITTA TORRONENa, MERJA KORKALAINEN b and SIRPA O KARENLAMPI b

aDepartment of Physzology and bDspartment of Bzochem~stry and Bwtechnology, Unwers,ty of Kuopw, P 0 Box 1627, SF-70211 Kuopw (F,nland) (Received November 25th, 1991) (Rewslon received March 27th, 1992) (Accepted March 31st, 1992)

SUMMARY

The mouse hepatoma cell hne Hepa-1 was shown to express an aldehyde dehydrogenase (ALDH) lsozyme whmh was reducible by TCDD and carcmogemc polycyclic aromatm hydrocarbons The induced actlwty could be detected with benzaldehyde as substrate and NADP as cofactor (B/NADP ALDH) As compared with rat hver and hepatoma cell hnes, the response was moderate (maxlmally 5-fold). There was an apparent correlation between this specific form of ALDH and aryl hydrocarbon hydroxylase (AHH) m the Hepa-1 wild-type cell hne -- m terms of mduclblhty by several chemmals However, the magmtude of the response was clearly smaller for ALDH than for AHH Southern blot analyms showed that a homologous gene (class 3 ALDH) was present m the rat and mouse genome The gene was also expressed m Hepa-1 and there was a good correlatmn between the increase of class 3 ALDH-speclfic mRNA and B/NADP ALDH enzyme activity after exposure of the Hepa-1 cells to TCDD It ~s concluded that class 3 ALDH is reducible by certain chemmals m the mouse hepatoma cell hne, although the respective enzyme is not reducible m mouse hver m wvo

Key words Aldehyde dehydrogenase -- Induction -- Mouse -- Hepa-1 -- Cell hne -- Hepatoma

INTRODUCTION

Aldehyde dehydrogenase (aldehyde:NAD ÷ oxldoreductase, EC 1 2 1 3, ALDH) occurs m most mammahan tissues, where it catalyses the oxidation of Correspondence to R TSrronen, Department of Phymology, Umverslty of Kuoplo, P O Box 1627, SF-70211 Kuoplo, Finland

0009-2797/92/$05 00 © 1992 Elsewer ScmntlfiC Pubhshers Ireland Ltd Printed and Pubhshed m Ireland

108

aldehydes to carboxyhc acids It thus has a detoxmatwe function There are several mozymes of ALDH. Induction of two distract lsozymes m rat hver is well documented Phenobarbital was first dmcovered to enhance ALDH actwlty [1] Mlrex and DDT are phenobarbital-type reducers, whmh mcrease cytoplasmm ALDH actlvaty charactemzed by preferentially catalysmg the metabohsm of propmnaldehyde or phenylacetaldehyde m the presence of NAD as cofactor [2] TCDD (2,3,7,8-tetrachlorodlbenzo-p-dmxm) [2-4], polychloranated blphenyls [2,5] and the polycychc aromatm hydrocarbons /~-naphthoflavone [2,6], 3methylcholanthrene [2,4-7], benzo[alpyrene [4-7], chrysene [4,5,7] and benz[a]anthracene [5,7] reduce another ALDH lsozyme, whmh differs from the phenobarbital-type enzyme, e g m its abdlty to oxidize benzaldehyde m the presence of NADP (B/NADP ALDH) The endogenous functmn of these reducible forms of ALDH is not known Bemdes rat hver, ALDH is reducible by phenobarbital and several polycyclic aromatm hydrocarbons also m primary cultures of rat hepatocytes [8-10] 3Methylcholanthrene and benzo[a]pyrene strongly reduce B/NADP ALDH actwlty also m the rat hepatoma cell hnes H4-II-EC3 and McA-RH7777 w~th mtermedmte or low basal actwatms [11] On the other hand, certmn rat hepatoma cell hnes express high constltutwe B/NADP ALDH actwlty whmh is only marganally reduced by these compounds [11,12]. In addltmn to a short-term exposure to varmus xenobmtms, also hepatocarcmogenesls mcreases B/NADP ALDH actwaty m the rat [13 - 15] The genes for the rat hver tumour-assocmted [16] and TCDD-mduclble [17] cytoplasmm ALDHs have been cloned and shown to be ldentmal [18] These ALDH lsozymes are referred to as class 3 ALDHs (ALDH3) according to the new nomenclature [191 As m the rat, B/NADP ALDH is also expressed m chemmally-mltmted mouse hver tumours [20,21]. However, in contrast to the rat, the mductmn of ALDH by TCDD or polycychc aromatm hydrocarbons has not been conwncmgly shown m the mouse [3,22] In the present report we descmbe the mductmn of B/NADP ALDH m the mouse hepatoma cell hne Hepa-1 after a short-term exposure to these chemmals Ewdence is also gaven to show that the reduced form of ALDH belongs to class 3 ALDHs MATERIALS AND METHODS Cell culture A subclone Hepa-lclc7 of the mouse hepatoma cell hne Hepa-1 [23] and the rat hepatoma cell line HTC [24] were obtamed from Dr D W Nebert, Unlvermty of Cincinnati Medmal Center, Cincinnati, Ohio The cells were grown as monolayers m a-MEM supplemented wath 5% foetal calf serum 0rradlated), 100 umts/ml pemcllhn and 0 1 mg/ml streptomycin The cell culture medmm, foetal calf serum and antlblotms were purchased from Glbco (Pmsley, UK) For ALDH enzyme actwlty and mRNA studies, 1 3 × 106 cells were plated on an 85-mm dmh m 10 ml of the medmm. The mducers were dissolved m dlmethyl sulphoxlde (DMSO) The final concentration of DMSO m the culture medmm never exceeded 0.5%

109

ALDH and A H H assays To assay ALDH or aryl hydrocarbon hydroxylase (AHH) the cells were harvested 24, 48 or 72 h after the initiation of exposure to the reducers Followmg removal of the medium, the cell surface was washed twine with 5 ml of cold Dulbecco's phosphate buffered sahne without calcmm or magnesmm (1% NaC1, pH 7 2) The cells were scraped and collected as pellets to be stored at - 8 0 ° C The cell pellets were thawed and somcated briefly m 1 ml of me-cold 33 mM potassmm phosphate buffer (pH 7 5) containing 0 25 M sucrose ALDH actlvltms were assayed at 37°C by momtormg the reduction of NAD(P) at 340 nm The assay mixture (final volume 1 ml) for non-specffic ALDH activity (P/NAD ALDH) contained 70 mM sodmm pyrophosphate buffer (pH 8 0), 1 mM pyrazole to mhlblt alcohol dehydrogenase, 1 mM NAD as cofactor and 5 mM propmnaldehyde (dissolved m water) as substrate For B/NADP ALDH actwlty 2 5 mM NADP was used as cofactor, 0 1 mM benzaldehyde (dissolved m methanol, final concentratmn 1%) as substrate and pyrazole was omitted The blanks contaming no aldehyde were run for every sample The actlvltms were calculated as nmol of NAD(P)H formed/mm per mg protein In ten different experiments carned out dunng a 1-year period, the B/NADP ALDH actlwty of DMSO-treated (control) Hepa-1 cell cultures ranged from 2 9 to 13 4 (mean 6 8) nmol NADPH mln- 1 mg protein - 1 The highest actlwtms were detected with freshly prepared benzaldehyde solutmns Therefore, some of the varmtmns m the basal B/NADP ALDH actlwtms can probably be explained by shght vanatmns in the assay condltmns However, despite the differences m the control levels, the relative response to the reducers was stable from one experiment to another AHH actlwty was assayed using a method onganally described by Nebert and Gelbom [25], wath some mo&flcatmns The method is based on the hydroxylatmn of benzo[a]pyrene to its 3-hydroxy derivative, whmh is measured fluorometrmally Cell suspensmns were incubated at 37°C for 15 mm in a mixture (final volume 1 ml) containing 25 mM potassium phosphate buffer (pH 7 5), 3 6 mM magnesmm chloride, 0 36 mM NADPH, 0 42 mM NADH, 675 #g of albumin and 80 ~M benzo[a]pyrene (dissolved in acetone, final concentratmn 5%) The reactmn was stopped by the addltmn of 1 ml of me-cold acetone and 3 25 ml of hexane After wgorous shaking (vortex) a 2 5-ml sample of the orgamc phase was taken and extracted with 2 ml of 1 M sodmm hydroxide The fluorescence of 3-OHbenzo[a]pyrene m the alkali phase was determined at the excltatmn and emlssmn wavelengths of 391 and 517 nm, respectively The assay was cahbrated with 3OH-benzo[a]pyrene and quinine sulphate The actlwty is expressed as pmol of 3OH-benzo[a]pyrene formed/mm per mg protein Protein was assayed by the method of Bradford [26] w~th Coomassm Brllhant Blue G-250 dye (Bm-Rad, Rmhmond, Cahforma) and using bovine serum albumin as standard Southern blot hybr~d~zatwn DNA was isolated from the cultured Hepa-1 and HTC cells using phenol extraction [27] Digestions were carried out under standard conditions Hybridization was performed with the rock-translated probe p12x p12x was a 1 150 bp cDNA clone specific for the TCDD-mduclble rat hver ALDH [17] and was a gift from Dr. T J. Dunn, McArdle Laboratory for Cancer Research, Umverslty of Wisconsin, Wisconsin

110

Analys~s of RNA RNA was isolated from the cultured cells as prewously descmbed [28] The purity of RNA was assessed by measunng the ratio between the absorbance readings at 260 nm and 280 nm The concentration of RNA was determined at 260 nm For slot blot hybrldmatmn analysm, 7 5 or 15 tzg of total RNA was diluted m 10 x SSC. The mLxture was applied on a nylon filter through a filtration manifold RNA was fixed by UV hght (254 nm, 5 mln) The filters were prehybradlzed for about 4 h at 42°C in 15 ml of a solution contammg 5 x Denhardt's solutmn, 6 x SSC, 0 5% SDS, 100 tzg/ml denatured salmon sperm DNA and 50% formamlde The hybm&zatmn mLxture also contmned 0 01 M EDTA and the pl2x probe In every experiment a duphcate filter was hybmdlzed to a cDNA probe for hamster CAD (carbamyl-P synthetase, aspartate transcarbamylase, dlhydroorotase) gene [29] CAD was obtamed from Dr W Lmo, Stanford Umverslty Medmal Center, Stanford, Cahforma and was used for the standardmatmn of the amount of ALDH mRNA bound to the filter during slot blot hybmdlzatmn analysm. The probes were rock-translated to a specific actwlty of - 3 x 108 counts/mm per/~g DNA and added to the hybmdlzatmn mixture The mRNA-DNA hybradlzatmn was camed out for 18-24 h at 42°C Following the hybmdlzatlon, the filters were washed three times with 2 x SSC plus 0 1% SDS at room temperature and twine w~th 1 x SSC plus 0 1% SDS at 60°C The filters were then autoradlographed (Kodak X-Omat AR-5 film) The relatwe amounts of ALDH mRNA were obtained by denmtometmc scanning (500 nm) of the X-ray films and by integrating the surface areas of the peaks RESULTS

Inductwn of ALDH by varwus chemtcals Hepa-1 mouse hepatoma cell culture was exposed to several compounds known to be reducers of either ALDH or the mmrosomal cytochrome P-450 system The concentrations of several compounds were chosen based on their ablhty to maximally induce AHH m Hepa-1 [28,30] The non-specific ALDH actwlty was assayed with proplonaldehyde as substrate and NAD as cofactor (P/NAD ALDH). Benzaldehyde and NADP were used to assay the ALDH form (B/NADP ALDH) whmh is inducible, e g by TCDD and carcmogemc polycychc aromatm hydroearbons m rat hver Among the ten chemicals tested, TCDD (1 nM), 3methylcholanthrene (1 /~M), benz[alanthracene (13 /~M) and chrysene (25 /~M) were able at least to double P/NAD ALDH actwlty (Table I) In addmon to these chemmals, B/NADP ALDH actwlty was also elevated by benzo[a]pyrene (1/~M), a-and/~-naphthoflavone (10/~M) and CPBT (100/~M). Under the condmons used, ehrysene, benz[a]anthracene and TCDD were the most potent reducers of ALDH aetwatles. Anthracene was not an mducer Mlrex (5-100/~M) and DDT (20 ~M) had no effect on either ALDH actwaty The time course of reduction of ALDHs m Hepa-1 were studied for TCDD (1 nM), 3-methyleholanthrene (1 /~M) and 3-naphthoflavone (10 /~M) Maximal aetlwtms were reached after a 1- or 2-day-exposure of the cells to 3-methylcholanthrene or/3-naphthoflavone and after a 2- or 3-day-exposure to TCDD

111 TABLE I EFFECTS OF VARIOUS COMPOUNDS ON ALDH ACTIVITIES IN HEPA-1 CELL LINE Chemmal a

PiNAD ALDH b

B/NADP ALDH b

Control (0 5%DMSO) TCDD(lnM) 3-Methylcholanthrene (1 #M) Benzo[a]pyrene (1/~M) Benz[a]anthracene (13 ~M) Chrysene(25/zM) Anthracene (100#M) c~-Naphthoflavone (10/~M) f~-Naphthoflavone (10/~M) Phenobarbltal(2mM) CPBT c(100/~M)

11 8 283 23 6 18 3 29 9 282 94 18 2 15 2 147 213

13 4 597 48 6 37 5 60 6 705 12 5 30 9 34 8 226 499

24 20 15 25 24 08 15 13 12 18

x x × × × x x × × ×

44 × 36 × 28 × 45 × 53 × 09 × 23 × 26 × 17 x 37×

aThe cells were exposed for 24 h to the compounds dissolved m DMSO The final concentrations are given in parentheses bThe specific actlwtms (nmol NAD(P)H × m l n - 1 × m g protein - 1) and the fold-reductions ( × ) compared with the controls are given Each value represents the mean of duphcate dishes of cells c2-(4'-chlorophenyl)benzothmzole

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AHH Fig 1 Correlation of B/NDP ALDH and AHH activities m Hepa-1 cell hne exposed to the compounds presented m Table I (except CPBT) for 24 h (AHH, pmol x mm -1 x mg protein -1) or 48 h (B/NADP ALDH, nmol NADPH x mm -1 x mg protein -1) Each point represents the mean of duphcate chshes of cells The abbrewatmns used are ANF, a-naphthoflavone, ANTH, anthracene, BA, benz[a]anthracene, BNF, fl-naphthoflavone, BP, benzo[a]pyrene, CHR, chrysene, DMSO, chmethyl sulphomde, 3-MC, 3-methylcholanthrene, PB, phenobarbital, TCDD, 2,3,7,8tetrachlorodlbenzo-p-dlomn

112

Correlatwn between ALDH and AHH tnductwn In Fig 1 the B / N A D P A L D H actlwtms are plotted a g m n s t the A H H actwltles for the compounds p r e s e n t e d m Table I (CPBT excluded). The best correlation b e t w e e n these enzyme activltms was d r a w n f r o m d a t a obtmned w h e n Hepa-1 cells w e r e exposed to these compounds for 24 h for A H H activity and for 48 h for A L D H actlwtms. The correlation coefficmnts w e r e 0 95 and 0 92 for P / N A D and B / N A D P A L D H actwltms, r e s p e c t w e l y

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CPBT (pM) Fig 2 Dose response of ALDH and AHH reduction by TCDD (A) and CPBT (B) m Hepa-1 cell hne The cells were exposed to the chemmals for 24 h The results are expressed as fold reduction calculated by gwmg the value 1 to DMSO-treated control cells Each point represents the mean of duphcate dishes of cells O, P/NAD ALDH, 0, B/NADP ALDH, A, AHH

113 The dose response curves for TCDD and CPBT mductmn of ALDH and AHH are presented m Fig 2. Maximal P/NAD ALDH, BfNADP ALDH and AHH actlwtms were obtained with 2 nM TCDD The maximal-fold mductmns were 1 7 for P/NAD ALDH, 4 7 for B/NADP ALDH and 35 for AHH Maximal P/NAD ALDH and B/NADP ALDH actlwtms were obtained with 60 ~M CPBT, the foldmductmns being 1 8 for PfNAD ALDH and 4 8 for B/NADP ALDH The maxxmal AHH actavxty reducible by CPBT was apparently not reached at the concentratmn range used With the highest CPBT concentratmn used (100 ~M), the fold-mductmn was as great as 102

Inductwn of ALDH by polycychc aromatw hydrocarbons of d~fferent carc~nogemc potency The non-carcmogemc polycychc aromatm hydrocarbons anthracene and phenanthrene did not increase either P/NAD ALDH or B/NADP ALDH actwltms at the concentratmn range of 5 - 1 0 0 #M The weak or moderate carcmogens chrysene and benz[a]anthracene increased both PfNAD ALDH and B/NADP ALDH (Fig 3) actwltms Maximal actwltes were reached with 10-25 t~M reducer Chrysene up to 50 ~M and benz[a]anthracene up to 25 tLM were not toxin to the cells The potent carcinogens benzo[a]pyrene and 3methylcholanthrene increased both PfNAD ALDH and B/NADP ALDH actwltes However, at concentrations higher than 5 #M the actiwtms started to decrease due to toxmlty of the compounds to the cells

30 C

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Concentration (I~M) Fig 3 Doseresponse of B/NADP ALDHmductmnby polycychcaromatichydrocarbonsm Hepa-1 cell hne The cells were exposed to various concentrations of benz[a]anthracene (BA), benzo[a]pyrene (BP), chrysene (CH) and 3-methylcholanthrene(3-MC) for 24 h Each pomt represents the mean of duphcate dishes of cells

114

Correlatwn of ALDH3 mRNA levels unth B/NADP ALDH enzyme aztw~t~es ,n Hepa-1 Southern blot analysis was conducted in order to find out whether DNA homologous to the rat TCDD-induclble class 3 ALDH-speclfic cDNA probe was present In Hepa-1 One or two bands for genomm DNAs digested with either EcoRI, BamHI, H~nd III or Pst I were observed for both Hepa-1 and HTC cell lines HTC was chosen as the positive control, because the cDNA probe used has

Control 1 nM 5 nM 20 nM 50 nM 100 nM

7

B.

6

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4 "0

2 1

0

20

40

60

80

100

TCDD (nM) Fig 4 (A) Slot blot analysis of ALDH3 mRNA m Hepa-1 cell hne (B) Dose-dependent mductmn of ALDH3 mRNA (O) and B/NADP ALDH actlwty (0) The cells were exposed to varmus concentratmns of TCDD for 24 h The results are expressed as fold reductions Each point represents the mean (± S E ) of three independent experiments

115

been shown to be ldentmal with ALDH3 m HTC [17] Only one band m the Southern blot strongly suggested that there was a very specific binding of the probe to only one gene This also allowed us to use the slot blot analysis instead of Northern analysis m the quantltatmn of class 3 ALDH-specffic mRNA m Hepa-1 In order to compare the amounts of total RNA bound to the filters, a duphcate filter was hybridized to a cDNA probe for hamster CAD gene [ 29] The results were similar with or without correctmg the data by CAD mRNA The time-dependent inductmn of ALDH3 mRNA and BfNADP ALDH actlwty m the Hepa-1 cell hne by TCDD showed that the maximal mRNA level was reached at 24 h The enzyme actlwty was delayed by several hours and the maximal actlwty was reached after 48 h The dose-dependent inductmn of ALDH3 mRNA and BfNADP ALDH activity correlated well (the correlatmn coefficmnt was 0 93). The maximal-fold mductmns were obtained with 5 - 20 nM TCDD (Fig 4) These data show that the amount of enzymatm activity is concordant with the specffic mRNA levels DISCUSSION

In the present study, an reducible BfNADP ALDH is described m the mouse hepatoma cell hne Hepa-1 A prehmmary report has been pubhshed earher [31] The mducers mcluded TCDD, carcmogemc polycychc aromatm hydrocarbons, aand 3-naphthoflavone and CPBT. With the exception of a-naphthoflavone and CPBT, these compounds are known reducers of the B/NADP ALDH type lsozyme m rat hver [2 - 7] A pecuhanty m the present observation is that, unhke m the rat, the normal mouse hver cytosohc P/NAD or BfNADP ALDH has not been found to be reducible by TCDD [3] or 3-methylcholanthrene [22] The reduction kinetics and magmtude of reduction of B/NADP ALDH seen m the mouse Hepa-1 cell hne was different from that prevmusly observed m rat hepatoma cell hnes. The fold-reduction by 3-methylcholanthrene or benzo[a] pyrene was 3-17-times smaller m Hepa-1 than that reported for the rat hepatoma cell hnes H4-II-EC3 or McA-RH7777 [11] It should be noted, however, that Lm et al [11] used very high reducer concentrations (1 mM) whmh according to our expermnces would have been extremely toxin to the cells In Hepa-1, max~mal reduction of BfNADP ALDH was seen after 1 - 2 days of exposure to 3methylcholanthrene, whereas m H4-II-EC3 it is not reached until after 4 days [111 In rat hver, the mductmn of B/NADP ALDH appears to correlate well with the carcmogemc potency of the polycychc aromatic hydrocarbon reducers [7] In Hepa-1, a similar correlatmn was not obwous No mductmn of ALDH by the noncarcmogemc polycychc aromatm hydrocarbons anthracene and phenanthrene was observed. However, the weak or moderate carcinogens chrysene and benz[a] anthracene gave maximal ALDH actlwtms as high or even higher than those obtamed by the potent carcinogens benzo[a]pyrene and 3-methylcholanthrene Maximal act~wtms were, however, reached by lower concentratmns of benzo[a]pyrene and 3-methylcholanthrene than of benz[a]anthracene and chrysene. A problem was that benzo[a]pyrene and 3-methylcholanthrene are

116

more or less toxin to the cells, mamfested as a rapid dechne of the actlvltms as the reducer concentration was mcreased This toxicity of the more potent carcmogens makes the comparison of the mduclng effects of the four polycychc aromatm hydrocarbons rather difficult In the present study, no induction of ALDH by Mlrex or DDT and only a marginal reduction by phenobarbital was observed m Hepa-1 Because Mlrex and DDT are phenobarbital-type reducers of ALDH [2], the results suggest that there is practmally no reduction of phenobarbital-type ALDH m Hepa-1 Associations between the reduction of ALDH and of the mmrosomal cytochrome P-450 system by phenobarbital [1,32-34] or TCDD [3,17] have been studmd m rats and mine, but no relationship has been found However, Nebert and coworkers suggest that ALDH may be under Ah receptor control [35] Hepa1 possesses an reducible AHH [28,30] and offers thus a model to study the associations There was a good correlation between AHH and B/NADP ALDH mduclblhty by various chemmals The only exception was CPBT which induced ALDH and AHH by 4- and over 100-fold, respectively, as compared with 4- and 45-fold by TCDD CPBT has been observed to Induce AHH exceptmnally strongly [30] However, the same does not seem to be true for B/NADP ALDH Furthermore, the apparent correlatmn between AHH and B/NADP ALDH detected in the present study is questioned by our prehmmary results with AHH-deficmnt mutant cell hnes of Hepa-1 whmh possess variable B/NADP ALDH actlwtms [31] Those results are in concordance with the results of Dunn et al [17] showlng a lack of coordination m the regulation of the TCDD-mduclble ALDH and cytochrome P-450c m the rat They observed that the induction of rat hver ALDH by TCDD differs from that of cytochrome P-450c (expressed as AHH) with regard to dose response relationship, reduction kinetics and tissue specificity However, the possibility that Ah locus has some role m the mductmn of ALDH (especially BfNADP ALDH) cannot be ruled out conclusively and addltmnal factors may be needed to explain the final responses In the rat, identity of the hver cytosohc ALDH induced by 3-methylcholanthrene, the tumour-assoclated ALDH and stomach ALDH has been suggested [36]. A very similar enzymatic species is found m normal rat urinary bladder [37] and cornea [38] A similar constitutive or reducible B/NADP ALDH is also present m certain rat hepatoma cell lines [11,12,39] As m the rat, B/NADP ALDH is expressed m chemically initiated mouse hver tumours [20,21] Among the several constitutive murme ALDHs known [40], AHD-4 which occurs in mouse stomach and eye tissue [41,42], can with certainty be referred to as B/NADP ALDH and is therefore also a possible candidate for an analogous enzyme to the reducible B/NADP ALDH m Hepa-1 cells Conclusive evidence on the identity between rat and mouse B/NADP ALDH cannot, however, be drawn on enzymatm basis, especially because mouse hver cytosohc B/NADP ALDH seems not, unhke the rat enzyme, to be reduced by TCDD [3] or 3methylcholanthrene [22] The best way to study relationships between different ALDH lsozymes is to work at the DNA and mRNA levels The cDNAs for the tumour-assocmted B/NADP ALDH (from the rat hepatoma cell line HTC) and TCDD-mduclble

117 B / N A D P A L D H (from r a t liver) a p p e a r to be ldentmal belonging to class 3 A L D H s [ 1 6 - 1 9 ] . In the p r e s e n t study, the identity of the putative analogous enzyme m the mouse Hepa-1 cells with these class 3 A L D H s was studied The clone p l 2 x isolated f r o m a c D N A library p r e p a r e d f r o m the h v e r of a TCDDinduced r a t [17], corresponding to the middle two-thirds of the full-length cDNA, was shown to detect specific D N A sequences m Hepa-1 Although there is a ten amino acid remdue s e g m e n t which is m v a r l a n t m all A L D H s t r u c t u r e s thus f a r known [18] (the nucleotlde sequences do not have a p e r f e c t match), the specffmlty of the p r o b e s for class 3 A L D H has also been p r o v e n in the r a t [16,17] the probes recogmze no sequences m normal or phenobarbital-reduced r a t h v e r A f u r t h e r ewdence for the identity of B f N A D P A L D H m Hepa-1 with r a t class 3 A L D H was t h a t t h e r e was a clear lnductmn of class 3 ALDH-speclfiC m R N A m Hepa-1 The time course of induction of the e n z y m e and the corresponding m R N A correlated well The s a m e good c o r r e l a t m n was also seen m the dose response curve The tumour-speclfic and stomach A L D H s e e m to be the same in the r a t and mouse The only clear difference b e t w e e n the r a t and mouse is the reducibility of A L D H by compounds like T C D D and 3-methylcholanthrene H o w does one then explain the reducible B / N A D P A L D H m the mouse h e p a t o m a cell hne Hepa1 w h e n the s a m e induction is not seen m mouse liver in vlvo~ The s y s t e m p r e s e n t e d here m a y offer an i n t e r e s t i n g model to study the r e g u l a t m n of class 3 ALDHs ACKNOWLEDGEMENTS The skillful techmcal assistance of Ms Ralsa Malm~vuon and Ms R n t t a V e n a l a m e n is gratefully acknowledged Mr H a r n Kokko is t h a n k e d for his help m some e x p e r i m e n t s REFERENCES

1 R A Deltnch, Genetm aspects of increase m rat hver aldehyde dehydrogenase reduced by phenobarbital, Scmnce, 173 (1971) 334-336 2 R A Deltnch, P Bludeau, M Roper and J Schmuck, Induction of aldehyde dehydrogenases, Blochem Pharmacol, 27 (1978) 2343-2347 3 R A Deltrmh, P Bludeau, T Stock and M Roper, Induction of different rat hver supernatant aldehyde dehydrogenases by phenobarbital and tetrachlorodlbenzo-p-&oxm, J Blol Chem, 252 (1977) 6169- 6176 4 M Marselos, R TorrSnen, T Kolvula and M Kolvusalo, Comparison of phenobarbital- and carcinogen-reduced aldehyde dehydrogenases In the rat, Blochlm Blophys Acta, 583 (1979) 110 - 118 5 M Marselos and V Vaslhou, Effect of varmus chemicals on the aldehyde dehydrogenase activity of the rat hver cytosol, Chem -Blol Interact, 79 (1991) 79-89 6 E Rltter and L C Enksson, Kmetms of reduction of cytosohc benzaldehyde NADP and proplonaldehyde NAD aldehyde dehydrogenase actlwtles m rat hvers from male Wlstar rats, Carcinogenesis, 12 (1991) 751- 758 7 R T6rr6nen, U Noummnen and 0 Hanmnen, Induction of aldehyde dehydrogenase by polycychc aromatm hydrocarbons m rats, Chem -Blol Interact, 36 (1981) 33-44

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M Marselos and G Mlchalopoulos, Phenobarbital enhances the aldehyde dehydrogenase activity of rat hepatocytes in wtro and in vlvo, Acta Pharmacol Tomcol, 59 (1986) 403- 409 M Marselos, S C Strom and G Mlchalopoulos, Enhancement of aldehyde dehydrogenase activity of human and rat hepatocyte cultures by 3-methylcholanthrene, Cell Biol Toxmol, 2 (1986) 257 - 269 M Huang and R Lmdahl, Effects of hepatocarcmogemc initiators on aldehyde dehydrogenase gene expression in cultured rat hepatic cells, Carcinogenesis, 11 (1990) 1059-1065 K -H Lm, A L Winters and R Lmdahl, Regulation of aldehyde dehydrogenase activity in five rat hepatoma cell lines, Cancer Res, 44 (1984) 5219-5226 K-H Lln, M F Leach, A L Winters and R Llndahl, Characteristics and aldehyde dehydrogenase actlwty of four rat hepatoma cell lines produced by diethylnitrosamlnephenobarbital treatment, In Vitro Cell Dev Biol, 22 (1986) 263-272 R Llndahl, S Evces and W-L Sheng, Expression of the tumor aldehyde dehydrogenase phenotype during 2-acetylaminofluorene-Induced rat hepatocarclnogenesls, Cancer Res, 42 (1982) 577- 582 S M Wlschusen, S Evces and R Llndahl, Changes in aldehyde dehydrogenase activity during dlethylmtrosamine- or 2-acetylaminofluorene-lmtiated rat hepatocarcinogenesis, Cancer Res, 43 (1983) 1710-1715 E Rltter and L C Enksson, Aldehyde dehydrogenase actlvmes in hepatocyte nodules and hepatocellular carcinomas from Wistar rats, Carcinogenesis, 6 (1985) 1683-1687 D E Jones, J r , M D Brennan, J Hempel and R Llndahl, Cloning and complete nucleotide sequence of a full-length cDNA encoding a catalytically functional tumor-associated aldehyde dehydrogenase, Proc Natl Acad Sci USA, 85 (1988)1782-1786 T J Dunn, R Lmdahl and H C Pitot, Differential gene expression in response to 2,3,7,8tetrachlorodibenzo-p-dioxm (TCDD) Noncoordinate regulation of a TCDD-mduced aldehyde dehydrogenase and cytochrome P-450c in the rat, J Biol Chem, 263 (1988) 10878-10886 J Hempel, K Harper and R Lindahl, Inducible (class 3) aldehyde dehydrogenase from rat hepatocellular carcinoma and 2,3,7,8-tetrachlorodibenzo-p-dioxm-treated hver distant relatmnship to the class 1 and 2 enzymes from mammalian liver cytosol/mltochondrla, Biochemistry, 28 (1989) 1160-1167 Anonymous, Nomenclature of mammalian aldehyde dehydrogenases, in H Welner and G T Flynn (eds), Enzymology and Molecular Biology of Carbonyl Metabolism 2 Aldehyde Dehydrogenase, Alcohol Dehydrogenase and Aldo-Keto Reductase, Alan R Liss, Inc, New York, 1989, pp XlX-XXl R E Richmond and M A Perelra, Aldehyde dehydrogenase as a marker for chemically initiated mouse liver tumours, Cancer Lett, 31 (1986) 205-211 R E Richmond, A B Deangelo, F B Daniel and R Lmdahl, Immunohistochemical detection of tumour-assocmted aldehyde dehydrogenase in formahn-fixed rat and mouse normal liver and hepatomas, Hlstochem J , 22 (1990) 526-529 V VasfllOU,R TorrSnen, M Malamas and M Marselos, Inducibility of liver cytosohc aldehyde dehydrogenase actiwty in varmus animal species, Comp Biochem Physml, 94C (1989) 671 - 675 H P Bernhard, G J Darhngton and F H Ruddle, Expression of liver phenotypes m cultured mouse hepatoma cells Synthesis and secretion of serum albumin, Dev Blol, 35 (1973) 83- 96 E B Thompson, G M Tomkms and J F Curran, Induction of tyrosine a-ketoglutarate transammase by steroid hormones in a newly established tissue culture cell hne, Proc Natl Acad Sci USA, 56 (1966) 296-303 D W Nebert and H V Gelboin, Substrate-lnducIble mmrosomal aryl hydroxylase in mammalian cell culture I Assay and properties of induced enzyme, J Bml Chem, 243 (1968) 6242-6249 M M Bradford, A rapid and sensitive method for the quantltation of microgram quantltms of protein utilizing the principle of protein-dye binding, Anal BIochem, 72 (1976) 248-254 J Sambrook, E F Frltsch and T Mamatm, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 S 0 Kf~renlampl,K Tuoml, M Korkalalnen and H Raunio, Inductmn of cytochrome P450IA1 in mouse hepatoma cells by several chemicals, Biochem Pharmacol, 38 (1989) 1517-1525

119 29 G M Wahl, R A Padgett and G R Stark, Gene amphficatlon causes overproduction of the first three enzymes of UMP synthesis m N4phosphonacetyl)-L-aspartate-res]stant hamster cells, J Blol Chem, 254 (1979) 8679-8689 30 S 0 K~enlampl, K TuomL M Korkalmnen and H Raumo, 2-(4'-Chlorophenyl)benzothlazole is a potent reducer of cytochrome P450IA1 m a human and a mouse cell hne Anomalous correlation between protein and mRNA reduction, Eur J Blochem, 181 (1989) 143-148 31 R TSrrSnen and S Kfixenlampl,Associations of aldehyde dehydrogenase with aryl hydrocarbon hydroxylase m mouse hepatoma cells, in I Schuster (ed), Cytochrome P450 Biochemistry and Blophymcs, Taylor and Francis, London, 1989, pp 519-522 32 R A Deltrlch, A C Colhns and V G Erwm, C-enetmmfluence upon phenobarbital-reduced mcrease m rat hver supernatant aldehyde dehydrogenase actlwty, J Blol Chem, 247 (1972) 7232 - 7236 33 M Marselos, Genetic vamatlon of drug-metabohzmg enzymes m the Wmtar rat, Acta Pharmacol Toxlcol, 39 (1976) 186-197 34 T J Dunn, A J Koleske, R Lmdahl and H C Pltot, Phenobarbital-inducible aldehyde dehydrogenase m the rat cDNA sequence and regulatmn of the mRNA by phenobarbital m responsive rats, J Bml Chem, 264 (1989) 13057-13065 35 D W Nebert, D D Petersen and A J Fornace, Cellular responses to oxidative stress the [Ah] gene battery as a parachgm, Environ Health Perspect, 88 (1990) 13-25 36 M Kolvusalo, M Aarnm, M Baumann and P Rautoma, NAD(P)-hnked aromatic aldehydes preferring cytoplasmm aldehyde dehydrogenases m the rat Constitutive and reducible forms m hver, lung, stomach and intestinal mucosa, m H Welner and G T Flynn (eds), Enzymology and Molecular Bmlogy of Carbonyl Metabohsm 2 Aldehyde Dehydrogenase, Alcohol Dehydrogenase and Aldo-Keto Reductase, Alan R Lms, Inc, New York, 1989, pp 19-33 37 R Lmdahl, Ident~ficatmn of hepatocarcmogenesm-assocmted aldehyde dehydrogenase m norreal rat umnary bladder, Cancer Res, 46 (1986) 2502-2506 38 S Evces and R Lmdahl, Charactemzatmn of rat cornea aldehyde dehydrogenase, Arch Blochem Bmphys, 274 (1989) 518-524 39 R Lmdahl, D W Baggett and A L Winters, Charactenzatmn of aldehyde dehydrogenase from HTC rat hepatoma cells, Bmchlm Blophys Acta, 843 (1985) 180-185 40 C L Manthey, G J Landkamer and N E Sladek, Identlficatmn of the mouse aldehyde dehydrogenases important m aldophosphamlde detoxlficatmn, Cancer Res, 50 (1990) 4991 - 5002 41 E M Algar and R S Holmes, Purlficatmn and propertms of mouse stomach aldehyde dehydrogenase Ewdence for a role in the oxldatmn of peroxlchC and aromatm aldehydes, Bmchlm Bmphys Acta, 995 (1989) 168-173 42 R S Holmes, R A Popp and J L VandeBerg, Genetics of ocular NAD ÷-dependent alcohol dehydrogenase and aldehyde dehydrogenase m the mouse evidence for genetm identity with stomach lsozymes and locahzatmn of Ahd-4 on chromosome 11 near trembler, Blochem Genet, 26 (1988) 191-205