Xenobiotic metabolism and mutation in a human lymphoblastoid cell line

Chem.-Biol.

Interactions,

Elsevier Scientific

53 (1985)

Publishers

Ireland

257

25’7-212

Ltd.

XENOBIOTIC METABOLISM AND MUTATION LYMPHOBLASTOID CELL LINE

CHARLES Program Institute

L. CRESPI, JOHN D. ALTMAN in

of

Toxicology, Technology,

Department Cambridge,

and MICHAEL

of MA

IN A HUMAN

A. MARLETTA*

Applied Biological 02139 (U.S.A.)

Sciences,

Massachusetts

(Received September 24th, 1984) (Revision received January 10th. 1985) (Accepted January 17th, 1985)

SUMMARY

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a] pyrene (B[a] P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a] P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a] P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a] P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B [a] P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of transdihydrodiol B[a] P metabolites in the metabolic profile. B[a] P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by a-naphthoflavone (ANF). *To whom correspondence should be addressed at: Room 56-229, MIT, Cambridge, MA 02139. Abbreviations: ABT, l-aminobenzotriazole; AHH, aryl hydrocarbon hydroxylase; AIA, allylisopropylamide; ANF, a-naphthoflavone (7,8-benzoflavone); B( a)P, benzo[ alpyrene; BNF, p-naphthoflavone (5,6-benzoflavone); hgprt, hypoxanthine guanine phosphoribosyl transferase; PAH, polycyclic aromatic hydrocarbon; TCDD, 2,3,7,8-tetrachlorodibenzop-dioxin. 0009-2797/85/$03.30

o 1985 Elsevier Scientific Publishers Printed and Published in Ireland

Ireland

Ltd.

258 words: Benzo[a]pyrene blast - Cytochrome P-450

Key

-

Xenobiotic

metabolism

- Human

lympho-

INTRODUCTION

The study of foreign compound metabolism and mutagenicity in continuous cell culture has been limited because many of the enzymes involved in these processes are not expressed during growth in continuous culture [1,2]. Most striking in this regard is the loss of the cytochrome P-450 family of isozymes in hepatocytes when grown in primary culture [l-3], and although recent studies have shown that it is possible to prevent the loss of oxidative activity for 24 h by modification of the culture media [4-6 1, the mechanism and utility of this phenomenon is unknown. Therefore, it has been necessary to carry out studies using whole-cell homogenates or subcellular fractions competent for this metabolic activity. However, a number of studies have shown that the spectrum of metabolites and DNA adducts formed after metabolism by subcellular fractions and intact cells differ significantly [7-g]; therefore intact cellular systems are desirable in the study of chemical mutagens. The enzymes involved in xenobiotic metabolism carry out four major types of reactions: oxidation, reduction, hydrolysis and conjugation. These reactions aid in the excretion of lipophilic foreign compounds [lo]. In some cases this metabolism generates chemically reactive and therefore potentially toxic and mutagenic products [ll] and current theories of chemical carcinogenesis rely on these metabolically generated reactive intermediates as the ultimate carcinogenic species [12]. The PAH represent well studied examples of compounds whose metabolites are chemically reactive, mutagenic and carcinogenic, with B[a J P serving as the prototype of this structurally diverse class of xenobiotics. The relationship of metabolism to cytotoxicity, mutagenicity and transformation is probably best understood for B[a]P [12] ; hence we have chosen B[a]P for our first characterization of the relationship between metabolism and mutagenicity in the AHH-1 cell line. The purpose of this paper is to report our initial biochemical characterization of the metabolically competent AHH-1 cell line. Specifically, we report here three specific characterizations and comparisons. These are: ( 1) A comparison of basal and induced activities using two substrates and two inhibitors of cytochrome P-450. (2) A comparison of the xenobiotic metabolism in this human lymphoblastoid cell line with xenobiotic metabolism in mitogen-activated peripheral human lymphocytes. (3) A characterization of B[a] P metabolism and mutagenicity in the AHH-1 cell line. MATERIALS

Chemicals

AND METHODS

were obtained

from the following

suppliers:

ANF,

/I-naphtho-

259

flavone (BNF) and B[a] P (Aldrich Chemical Co., Milwaukee, WI); NADPH, NADH, dibenz[a,c]anthracene dibenz[a,h]anthracene, fl-glucuronidase, aryl sulfatase, d-saccharic acid-( 1,4)-lactone (Sigma Chemical Co., St. Louis, MO); Arochlor 1254 (Analabs, Inc., North Haven, CT). [‘H]B[a]P and [‘4C]UDP glucuronic acid were obtained from New England Nuclear (Boston, MA). Authentic B[a]P metabolites: 3-hydroxy B[a]P, 9-hydroxy B[a]P, trans7,8dihydroxy-7,8dihydro B[a] P, trans-4,5-dihydroxy-4,5-dihydro B[a] P, trans-9,lOdihydroxy-9,10dihydro B[a] P-3,6B[al P, B[a] P-1,6dione, dione, B[a]P-6,12-dione and [3H]B[a]P-4,5-oxide were kindly provided by the National Cancer Institute Chemical Carcinogen Repository. 7-Ethoxyresorufin and resorufin were the generous gifts of Professor C. Walsh and Alan Klotz of MIT and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was a gift from Dr. William Greenlee of the Chemical Industry Institute of Toxicology (Research Triangle Park, NC). Allylisopropylamide (AIA) was a gift from Dr. W.E. Scott of Hoffman-LaRoche Inc. (Nutley, NJ). ABT was synthesized using published procedures [13] . Cell culture media and sera were obtained from either Flow Laboratories (McLean, VA) or Gibco Laboratories (Grand Island, NY). Cell culture

The isolation of the AHH-1 described elsewhere [ 141. B[a] P hydroxylase

cell line

and cell culture

procedures

are

assay

B[a] P hydroxylase activity was measured by the production of fluorescent phenolic metabolites of B[a] P [15] as modified for use with human lymphocytes [17]. 7-Ethoxyresorufin

deethylase

assay

Human lymphoblasts were prepared as for a B[a] P hydroxylase assay. The substrate was added to a 2-ml cell suspension in 2 ~1 dimethylsulfoxide (DMSO). Production of resorufin was measured by fluorescence. The cell suspension was centrifuged (1500 X g, 30 s) and the fluorescence of the supematant measured with excitation at 570 nm and emission at 586 nm. The centrifugation step was necessary to eliminate interferences due to light scatter. Resorufin concentration was determined by comparison to a resorufin standard. Epoxide

hydrolase

assay

Epoxide hydrolase activity was measured utilizing substrate according to the method of Glatt et al. [16]. UDP-glucuronyl

transferase

B[a] P-4,5-oxide

as

activity

AHH-1 cells (1 X 107) were centrifuged and resuspended in 0.5 ml of 0.1 M KH,PO, (pH 7.5). The cells were then freeze fractured and the substrate added in 2 ~1 of DMSO. Final substrate concentrations were: p-

260 nitrophenol (140 PM) and 3-hydroxy B[a] P and 9-hydroxy B[a] P (10 PM). The reaction was initiated by the addition of [ 14C] UDP glucuronic acid in 5 ~1 of water (0.5 pmol, spec. act., 0.4 mCi/mmol). The incubation was carried out at 37°C for 20 min. The mixture was then applied to an Amberlite XAD-2 column (1 X 10 cm) and washed with 20 ml of water to separate the unreacted [ 14C] UDP glucuronic acid from the conjugates formed. The conjugates were eluted with 20 ml methanol. Rat liver microsomes (0.25 mg protein) and boiled cells were used in the incubation as controls. Analysis of B[a]P metabolites AHH-1 cells were incubated in the presence of 25 ,uM purified [ 3H] B[a] P (spec. act., 500 mCi/mmol) for 45 min. The cell suspension was extracted three times with 2 ml of ethyl acetate. The combined extracts were dried with anhydrous magnesium sulfate, filtered and the ethyl acetate evaporated under a stream of nitrogen. The extract was dissolved in a small volume of ethyl acetate and the unmetabolized B[a]P separated from the metabolites on a silica gel G TLC plate using a benzene mobile phase. The metabolites were eluted from the silica gel with methanol, the silica gel separated by centrifugation and the methanol evaporated under nitrogen. Identical treatment of B[a]P metabolite standards showed no significant change during this isolation procedure. The metabolites were dissolved in 0.4 ml of methanol and the methanol concentration adjusted to 60% with water and unlabeled metabolite standards added. Metabolites were separated using a Micromeritics model 7000 high performance liquid chromatograph equipped with an Ultrasphere ODS (4.6 X 250 mm) reverse phase column (Altex, Inc., Berkeley, CA). Methanol concentration was initially 60% and increased linearly to 100% methanol over 40 min. A constant flow rate of 1.00 ml/min was maintained. Absorbance at 254 nm was monitored using a Micromeritics model 786 detector. Fractions (0.3 min/fraction) were collected, 10 ml of aquasol added per vial and radioactivity was measured using a Beckman model LS-3150 P liquid scintillation counter. Enzymatic hydrolysis of B[a]P metabolite conjugates AHH-1 cells were prepared as described above for B[a]P metabolism studies or the B[a]P hydroxylase assay. After incubation, 0.1 ml of 0.5 M acetic acid was added (final pH 4.5). Then 10 000 units p-glucuronidase or 10 units aryl sulfatase with 10 mM d-saccharic-(1,4)-lactone were added in 50 ~1 of 50 mM sodium acetate (pH 4.5). Acetate buffer only was added to control incubations. The mixture was incubated at 37°C for 10 min and the metabolite extraction and analysis performed as described earlier. Suicide inhibition assay Uninduced and BNF-induced AHH-1 cells were centrifuged and washed twice with RPM1 medium 1640 plus 5% (v/v) horse serum then resuspended

261

in the above medium either AIA’ (7.5 nM) were then centrifuged Gene-locus

mutation

and incubated for 3 h in the absence or presence of or 1-aminobenzotriazole (ABT) (2.24 nM). The cells and 7-ethoxyresorufin deethylase activity measured. assay

The procedures for measuring gene-locus mutations in AHH-1 cells have been reported previously [14] and were used here with the following modification. AHH-1 cells were pretreated for 24 h with 10 PM BNF to induce cellular mixed function oxidase activity. Cells were centrifuged, washed to remove the BNF, centrifuged and resuspended at 5 X lo6 cells/ml in RPM1 medium 1640 supplemented with 5% (v/v) horse serum and containing 1.5 mg/ml NADPH and 1 mg/ml NADH. AHH-1 cells were aliquotted into 25 cm2 tissue culture flasks (3 X 10’ cells/culture) and B[a]P and ANF added in DMSO (final DMSO concentration 0.1%). Cells were incubated for 1.5 h at 37°C. After incubation, 1 ml of the cell suspension was removed and extracted to determine B[a]P phenol production and the rest of the culture was centrifuged, resuspended to fresh medium at 3 X lo5 cells/ml and cultured to allow phenotypic expression of the induced mutations. RESULTS

Inducibility

of cellular

AHH

activity

The compounds in Table I have been tested for their ability to induce AHH activity in AHH-1 cells. Two indices of inducing ability have been calculated: one, fold induction over uninduced controls assayed under identical conditions, and two, induction relative to B [a] P-induced cultures. We have found that the fold induction varies depending on parameters such as cell doubling time during induction (shorter doubling times yield higher ind.uced AHH activities). The second index, the relative inducing efficiency compared to a simultaneous B[a] P-induced control yields more consistent results on independent days since both experimental and B[a]P controls are subject to the same influences. The AHH activity of AHH-1 cells was induced 6-20-fold by pretreatment with PAH inducers of cytochrome P-450 activity. B[a]P, dibenz[a,c] anthracene, dibenz[a,h]anthracene, benz[a]anthracene, 3-methylcholanthrene, TCDD and BNF were found to be potent inducers of AHH activity. Several other PAH were found to be weak inducers (a 2-4-fold induction): chrysene, 7,12-dimethylbenz[a] anthracene and perylene. Arochlor 1254 and phenobarbital, two hepatic inducers of cytochrome P-450, did not induce B[a] P hydroxylase activity in AHH-1 cells. The concentration-response for B[a] P induction of B[a] P hydroxylase activity was examined: a significant induction of activity was observed after a 24-h treatment with 1 X 1Om8 M B[a]P and a monotonic increase was observed to an apparent plateau at concentrations greater than 1 X lo-’ M. The induction of B[a] P hydroxylase activity occurred rapidly in AHH-1

262 TABLE

I

INDUCERS n.a., Not untreated

OF

AHH

ACTIVITY

applicable. The control cultures.

AHH n.d.,

activity was not done.

Compound

Range cont.

not

statistically

different

from

Induction relative

Fold induction

(PM)

the

level

in

to

B[alP Arochlor 1254 Benz [ a ] ant hracene

BlalP BNF Chrysene Dibenz[a,c)anthracene Dibenz[a,h]anthracene 7,l2-Dimethylbenz[a]anthracene Fluoranthene 3-Methylcholanthrene Perylene Phenanthrene Phenobarbital Pyrene TCDD Triphenylene

3-30 3-30 10 3-30 l-10 3-30 l-10 l-10 l-30 10 l-10 0.1-10 1 mM 0.1-10 0.1 0.1-10

pg/ml

1.3 14.2 13.8 11.4 3.7 20.1 13.6 2.1 1.0 6.9 2.7 0.8 1.2 1.0 14.2 0.7

0.07 1.03 1.00 0.82 0.37 1.45 1.5 0.2 n.a. 1.2 0.27 n.a. na. na. n.d. na.

cells. Induction was observed 1 h after exposure to B[a] P. B[a] P hydroxylase activity increased linearly with time, reached a plateau at 24 h and remained stable (in the presence of the inducer) for up to 72 h. A 24-h treatment was chosen for routine induction of cytochrome P-450 activity in AHH-1 cells. Comparison

of basal and induced

mixed

function

oxidase

activities

The kinetic parameters for two cytochrome P-450-associated activities, B [a] P hydroxylation and 7-ethoxyresorufin deethylation, have been determined. The apparent Km and V,, for the basal and induced (Fig. 1) B[a] P hydroxylase activities were 2.1 X 10e6 M, 0.07 pmol/106 cells/min and 8.6 X low6 M, 2.2 pmol/106 cells/min, respectively. The apparent Km and V max for basal and induced (Fig. 2) 7-ethoxyresorufin deethylase activities were 4 X lo-’ M, 0.07 pmol/106 cells/min and 1 X 10m6 M, 1 pmol/106 cells/ min, respectively. The kinetics of ANF inhibition of 7-ethoxyresorufin deethylase activity were examined. ANF was found to be a competitive inhibitor of the basal activity (Ki = 3 X 10e9 M) (Fig. 2A) and a linear mixed inhibitor of the induced activity (Ki = 3 X 10e7 M) (Fig. 2B).

263

(A)

INDUCED

I 0 --

pmolo

-12

30HBP

-8

-4

8

12

16

40

60

80

UNINDUCED

(B)

40

pmole

v

4

--

3OHBP

lo*

I

-60

I

-40

-20

BENZO(APYRENE

20

’ ~0-4(mo~or-‘)

Fig. 1. Kinetic analysis of B[n]P hydroxylase activity. Uninduced anthracene-induced (A) AHH-1 cells were incubated in the presence trations of B[a]P. Cell concentration was 8 x lo6 cells/ml.

(B) or dibenz[u,c]of different concen-

The apparent kinetic constants in cell culture systems are dependent on cell density in that the actual intracellular concentration of B[a]P or 7ethoxyresorufin will vary in relation to cell density. Our experiments were carried out at the same cell density and therefore the kinetic parameters can be directly compared. However, the constants are apparent in that the actual intracellular concentrations of the substrates are unknown. In contrast to the above observations, no difference between inactivation of the basal and induced ‘I-ethoxyresorufin deethylase activities by two suicide substrates, AIA and ABT, was observed (Table II). Cellular B[a]P metabolite production The cellular metabolite profile was examined by HPLC separation. Phenolic metabolites were the major products of oxidation (Fig. 3). When these phenols were further resolved, three components were observed

264

(A)

INDUCED 50

-5

--

0

5

IO

I 7-ETHOXYRESORUFIN

‘I0

UNINDUCED

-25

-20

-15

-10 I 7-ETHOXYRESORUFIN

15

5.

-5

20

-5hlolar-‘)

__

I 5

0 ‘1’

I IO

I 15

I 20

-6holor-‘)

Fig. 2. Kinetic analysis of 7-ethoxyresorufin deethylase activity. A: dibenz[a,c]anthracene-induced AHH-1 cells were incubated with different concentrations of ethoxyresorufin and the production of resorufin monitored by fluorescence (open squares). The addition of 3 x 10.’ M (open circles) and 1 x 10e6 M (closed circles) ANF to the incubation mixture results in inhibition of the deethylase activity. ANF was a linearmixed type of inhibitor. Cell concentration was 8 x lo6 cells/ml. B: uninduced AHH-1 cells were incubated with different concentrations of ethoxyresorufin and the production of resorufin monitored by fluorescence (open symbols). The addition of 1 x 1Om8 M ANF to the incubation mixture resulted in inhibition of the deethylase activity (closed symbols). This inhibition was competitive. Cell concentration was 8 x lo6 cells/ml.

(Fig. 4). The major component cochromatographed with the 9-hydroxy B[a]P standard, the second component is as yet unidentified, and the third component (the small shoulder in the trailing edge of the second peak) cochromatographs with 3-hydroxy B[a]P standard. Quinones were also produced; these quinones cochromatographed with B[a] P-1,6-dione, B[a] P-3,6-dione and B[a] P-6,lBdione standards. Two minor unidentified metabolites were observed. These were an early eluting peak (6-min reten-

265 TABLE

II

SUICIDE DEETHYLASE Cell

INHIBITION ACTIVITY

type

Uninduced

BNF-Induced

aAll values day.

represent

OF

BASAL

AND

BNF-INDUCED

Inhibitor

% Deethylase

None AIA ABT None AIA ABT

100 c 10 56a 15 100 f 4 61 14

the

mean

of 3 independent

7-ETHOXYRESORUFIN

activity

determinations

performed

on a single

tion time) and a peak which chromatographed between the trans-9,10dihydrodiol B[a] P and trans-4,5dihydrodiol B[a]P standards (18-min retention time). No transdihydrodiol B [a] P metabolites were observed. This observation is consistent with our observation that no epoxide hydrolase activity (using B[a] P-4,5-oxide as substrate) is detectable in AHH-1 cells. The extent of fl-glucuronidation and sulfation of B[a] P metabolites produced by induced AHH-1 cells was examined by enzymatic hydrolysis. Hydrolysis of the metabolite mixture in the B[a] P hydroxylase assay prior to extraction revealed a 12% increase in fluorescence after p-glucuronidase treatment (P< 0.05) and no change in fluorescence after sulfatase treatment. These measurements were extended by analysis of individual metabolite species. fl-Glucuronidase treatment preferentially released 3-hydroxy B[a] P (Fig. 3). Direct measurement of glucuronyl transferase activity with three different substrates, p-nitrophenol, 3-hydroxy B[a] P and 9-hydroxy B[a] P showed that the activity in AHH-1 cells was not detectable (less than 0.1 pmol/106 cells/min). Positive controls with BNF-induced rat liver microsomes yielded activities for 3-hydroxy B[a] P, 9-hydroxy B[a]P and pnitrophenol of 1.3, 0.8 and 1.4 nmol/mg protein/min, respectively. B[a]P metabolism and mutagenicity Production of fluorescent phenolic metabolites from B[a] P was monitored concurrently with the measurement of gene-locus mutation induction in BNF-preinduced AHH-1 cells. The amount of phenol production during treatment was linearly related to the frequency of induced mutations at the hgprt locus (Table III). ANF, an inhibitor of cytochrome P-450 activity, inhibited the induction of mutation by B[a] P. Linear regression analysis of the observed mutant frequency versus the amount of phenol production indicates that 6 pmole of B[a] P are metabolized per mutant induced.

266

t n

tt t

tt

40 INDUCED

‘0

E

30

,”

20 IO 0

(B) g-

30

0

-

20

INDUCED + _ DECONJUGATION

E ,”

IO 0

c) ‘0

(Cl 20

- BOILED

5 E a D

IO 0

B RETENTION

TIME

bin)

Fig. 3. B[o]P metabolites produced by dibenz[a,c]anthracene-preinduced AHH-1 cell B[a]P metabolites were produced and analyzed as described and Methods. The predominant metabolites produced were B[a]P phenols. TABLE

AHH-1 cells. in Materials

III

RELATIONSHIP

BETWEEN

Linear

analysis:

regression

B[o]P

METABOLISM

AND MUTAGENICITY

Y = 6.0 X - 9.9; r = 0.989.

Conditions

Negative control 10 jiM B[o]P 20 /.iM B[o]P 20 PM B[o]P + 0.1 /JM ANF 20 PM B[n]P + 1 /.iM ANF 1 crM ANF

X Mutant fraction mean f S.E.M.a 1.9 6.1 9.3 6.7 5.0 1.7

+- 0.5 zk 1.6 Ii 1.7 + 2.1 + 0.5 + 0.1

(X 106)

Y B[n]P metabolism mean f S.E.M.= 29 43 34 19 -

+1 +1 I! 2 +1

aB[a]P metabolism expressed as pmol of 3-hydroxy B[o]P equivalents per lo6 cells. Values represent the mean of 2 independent experiments each performed in duplicate cultures.

267 600

200

0

60 65 70 RETENTION TIME (minutes 1

Fig. 4. Separation of B[a]P phenols. B[a]P metabolites were prepared as described in Materials and Methods. Separation of the metabolites was achieved using an ultrasphere ODS column (4.6 x 250 mm), flow rate 1.00 ml/min, solvent composition was a linear gradient from 60% methanol/40% water to 100% methanol, 0.25% per min.

DISCUSSION

Aspects of AHH-1 cell xenobiotic metabolism resembled human peripheral lymphocyte metabolism, particularly with regard to the spectrum of inducers, the observed kinetic parameters for B[a]P hydroxylase activity and 7-ethoxyresorufin deethylase activity, and the predominant metabolism of B[a]P to phenols. The spectrum of inducers of B[a] P hydroxylase activity in AHH-1 cells is similar to that observed in mitogen-activated peripheral human lymphocytes. In both cell types various PAH are active while phenobarbital is inactive [17,18]. The apparent Km -values for B[a] P hydroxylation of mitogen-activated human lymphocytes have been reported to be 2-3 PM and 4-8 FM for the basal and 3-methylcholanthrene-induced activities respectively (range of three individuals); the basal and 3-methylcholanthrene-induced apparent V,,,-values were 0.02-0.03 pmol/106 cells/min and 0.05-0.33 pmol/106 cells/min respectively [ 171. Similar measurements in AHH-1 cells revealed that the apparent Km-values for B[a] P hydroxylation were 2.1 PM and 8.6 PM for the basal and dibenz[a,c]anthracene-induced activities respectively. The overall level of B[a]P hydroxylase activity in AHH-1 cells was somewhat higher than freshly isolated lymphocytes. No detailed kinetic data are available for 7-ethoxyresorufin deethylase activity in human lymphocytes. The level of 7-ethoxyresorufin deethylase activity observed in induced AHH-1 cells is comparable to that observed in 3-methylcholanthreneinduced human lymphocytes [ 191.

268 AHH-1 cells metabolize B[a]P predominantly to phenols but also to quinones. Human lymphocyte metabolism produces predominantly phenols but also produces quinones and dihydrodiols [20-241. No dihydrodiol metabolites of B[a]P were detected in AHH-1 cell incubations. This lack of dihydrodiol metabolite production in AHH-1 cells is apparently due to the absence of epoxide hydrolase activity. Freshly isolated human lymphocytes, however, have been reported to have significant levels of epoxide hydrolase activity [16]. The amount of epoxide hydrolase activity is the only major difference between AHH-1 cells and mitogen-activated human lymphocytes discovered in this investigation. Preliminary observations indicate that treatment of AHH-1 cells with 5-azacytidine causes a transient expression of epoxide hydrolase activity (unpublished observations). Additional manipulations may stabilize this expression and restore this important metabolic activity. The substrate specificities of the cytochrome P-450 isozymes present in lymphocytes and AHH-1 cells seem to differ from those of P-450 isozymes in human liver. While B[a] P hydroxylase activity is found in both liver and lymphocytes, Guengerich and coworkers did not find 7-ethoxyresorufin deethylase activity in human liver microsomes or in six cytochrome P-450 isozymes purified from those microsomes [25]. The significance of this difference is unknown, but as various purifications and immunological characterizations proceed, we will eventually be able to relate AHH-1 cytochrome P-450 isozymes to other human and animal P-450’s. A first step toward differentiating the characteristics of basal and induced activities was through kinetic measurements. Significant differences between the Michaelis constants, maximal velocities and inhibition patterns were found. Although our main purpose in performing the kinetic characterization was to compare kinetic parameters obtained from AHH-1 cells to the wealth of data from freshly isolated human lymphocytes, we were also curious as to whether the basal and induced activities represented different forms of the enzyme. Our attempts to differentiate between basal and induced 7-ethoxyresorufin deethylase activities by using suicide inhibitors of cytochrome P-450 revealed that both activities were equally inhibited by these catalytic-type inhibitors. Characterizations of these inhibitors with microsomes and purified isozymes have shown that differential isozyme reactivity is possible; however, overlapping inhibition is possible [26,27] , and may have occurred here. Therefore, at this point we can not conclude that the basal and induced activities represent different P-450 isoenzymes. The apparent differences in both Michaelis constants and maximal velocities between the basal and induced cytochrome P-450 activities is interesting given our previous observations of the mutagenic activity of xenobiotics in this cell line [14]. Four chemicals, lasiocarpine, 1-methylphenanthrene, aflatoxin-B, and dimethylnitrosamine, were tested for mutagenic activity in both uninduced and BNF-preinduced cells. These four chemicals were

269 equally mutagenic (<2-fold difference on a concentration basis) in uninduced and BNF-preinduced AHH-1 cells. Given that BNF preinduction increases the levels of two cytochrome P-450 associated activities, B[a]P hydroxylase and 7-ethoxyresorufin deethylase, about lo-fold, the <2-fold difference in mutagenicity was unexpected. These observations indicate that for these four chemicals, the BNF-induced catalytic activities do not comprise the rate limiting step in the induction of mutagenesis. Also, the lower, basal level of activity may be more important for the mutagenicity of these chemicals. In contrast to the four chemicals discussed above, a substantial difference in B[a] P mutagenicity was observed between uninduced and BNF preinduced cells. In this report a 1.5-h exposure of BNF preinduced AHH-1 cells to 10 PM B[a]P induced a 6-thioguanine resistant fraction of 4.2 X 10m6, which is 2.8 X 10e6 mutants induced/cell/h of exposure; in our previous report, a 24-h exposure of cells which were not BNF-preinduced to 10 PM B[a]P yielded a mutant fraction of 13 X 10-6, which is 0.5 X 10m6 mutants/ cell/h of exposure [ 141. Therefore induced cells are about 5-fold more sensitive. However, during the 24-h exposure, B[a] P induces the cellular capacity to metabolize B[a] P, therefore if the amount of B[a] P metabolism is the rate limiting step in the production of mutations under these conditions, uninduced cells are probably even less sensitive to B[a]P-induced mutation than the 24-h exposure data indicate. Our experiments examining the influence of ANF on the amount of metabolism and the induction of gene-locus mutations by B[a] P demonstrate that the induction of gene mutations in AHH-1 cells can be correlated with the metabolic activity of the cell, in particular with B[a]P phenol production. The inhibition of B[a]P metabolism and mutagenicity by ANF demonstrates the usefulness of this system for studying the influence of metabolism on the processes of mutation through the use of inhibitors. B[a] P-induced mutagenicity was linearly related to the amount of phenol production during B[a]P exposure and we have estimated the amount of B[a]P metabolism per mutant induced. Further characterization of this celI line and the mechanism of B[a]P-induced mutagenicity not involving dihydrodiol metabolites should yield a clearer understanding of alternate pathways of activation. AHH-1 cells will serve as a useful probe of the biochemical reactions involved in xenobiotic metabolism and their relationship to chemical mutagenesis because of the following attributes: (1) Several aspects of foreign compound metabolism in AHH-1 cells resemble those reported for human lymphocytes therefore AHH-1 cells have potential as a model of some aspects of human foreign compound metabolism. (2) AHH-1 cells grow readily in culture and form colonies with high efficiency; this will facilitate the isolation of variants with altered xenobiotic capacities. (3) A gene-locus mutation assay has been developed in this cell line which will allow the

270 determination of the influence of various metabolic pathways on the induction of gene mutations. The influence of alterations in xenobiotic metabolism, either by external agents such as inhibitors or through the selection of isogenic variants, on the susceptibility of the intact cell to the induction of gene mutations can be easily studied. (4) The parent ce!! line will allow for the unique characterization of B[a]P-induced mutagenicity where dihydrodiol metabolites are not involved. ACKNOWLEDGEMENTS

We are grateful to Sheila Collins for carrying out and 3-methylcholanthrene and to Dr. William G. discussions. This investigation was supported by ES00597, the Whitaker Health Science Fund of #EE77-S-02-4267 and DEA-C02-EY04267. M.A.M. Mitsui Career Development Assistant Professorship.

the studies with TCDD Thilly for his helpful NIEHS award #5-POlMIT and DOE award is the recipient of a

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