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Induction of aryl hydrocarbon hydroxylase in human peripheral blood lymphocytes by chrysene
CancerLet?crs, , ~1979) 313--318
313
© KSsevier/North-HollandScientificPublishersLtd.
INDUCTION OF ARYL HYDROCARBON HYDROXYLASE IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES BY CHRYSENE
D.R. SNODGRASSI'2, T.L. McLEMORE1'3"4, M.V. MARSHALL1, N.P. WRAYs'4, E.T. CANTRELLs, D.L. BUSBEE2 and M.A. ARNOTT1 1Department of Environmental Biology, U.T M.D. Anderson Hospital a~d Tumor Institute, Houston, TX 77025, 2Department of Bmlogicol Sczences, North Texas State University, Denton, TX 76201, sZ 2partment of Pharmacology, Texas College of Osteopathze Medzcm~, Ft. Worth, TX, 3Department of Medwme, Baylor College of Medicine, Houston, TX 77025, 4Veterans Admin~strmwn Hospital, Houston, TX 77211 (U.S.A )
(Reeewed 3 May 1979) (Accepted 4 June 1979j
SUMMARY Many of the polycyclic vromatic hydrocarbons (e.g., benzo[a]pyrene (B[a] P), benzanthmcene (B A), 3-methylcholanthrene (3-MC)) are not only carcino,~enic, but also induce AHH in human tissue~. Recently, chryse~e has been implicated as an etiolo~,dc determinant of chemical carcinogenesis. Here we describe the ability of ehrysene to induce ~HH in cultured human lymphoey~es. Lymphoeytes were obtained from 9 healthy subjects, divided into 2 sets, and cultured in duplicate, triplicate, or quadruplicate for 48 h. Chrysene (25 #M final concentration) in acetone was then added to the induced[ culture set and the control set received acetone alone. Lymphocytes were then cultured ar~additkma} T24 h before harvest;ing. AHH was quantitsted by a fluorometric analysis of the phenolic metabolites produced by incubating tl~e [ymphocytes with B[a]P for 35 rain. A sigrdfieant increase in enzyme induction occurred in the chlrysene-induced cultures compared with control (non-induced) cells (one-tailed student t-test; P < 0.001). It was also observed that the interindividual vm'iation in AHH £uducibflity seen with otl~er PAHs is also observed with chrysene. IN'I~ODUCrION Aryl hydrocarbon hydroxylase ( A ~ ) is a membrane bound monooxygenase :responsible for the metabo]~ism of a wide variety of hydrophobic Address all correspondence to. Don Snodg~ss, c/o General MedicineSection (111C),
Vete~m'J~AdministrationMedical Center, 2002 Holcomb Blvd., Houston, TX 77211, U.S.A.
314 I00
1"--7Control L._J Chrysene induced
80 Achwty (milliunds) 40
A
C
D
E
F
G
H
I
lndividuols
Fig. 1. Structural comparisonbetween chlTseneand commonly used aryl hydrocarbon hydroxylase (AHH) inducers.
xenobiotics,includi~qgchemical carcinogens,to intormediate products with enhanced mutagenic and carcinogenicpotential.This enzyme complex has been implicated in the etiology of lung cancer in ,.~xparimentalanimals [1,17,18] and man [7,8,11].A H H isinduciblein human tissuesby ~arious PAHs such as benzanthmcene 3-MC, B[a]P, dimethylbenzanthmcene and tetrachlorodibenzo-p-dioxin[9,12,16]. In the present report, we have investigatedthe role of chrysene (which is similarin structureto other commonly used inducers (Fig.I) and isan environmental contaminant formed as a consequence of pyrocondensation processes,such as the combustion of tobacco products [3 ])as an inducer of A H H in cultt'Lredhuman lymphocytes. MATERIALS AND METHODS
Lymphocyte i~olation and culture techniques Venous blood (60 ml) was obtained from volunt,~ers who were not receiving medication that might he expected to alter the AHH activity of their cells. The blood was diluted 1 : i with heparinized sahne (10 units heparin/ ml saline) and mononuclear cells were separated I~.y Ficoll-Hypaque sedimentation as previously described [4]. Cells were removed from the hypaqueplasma interface and cultured by the method of McLemore et al. [13]. Two sets of cultures were incubated in either duplicate, triplicate or quadruplicate (depending on the number of cells obtained from a particular individual). One set contained cultures to which 25 #M chrysene {ICN, Irvine, CA) in 40 #l acetone had been added after 48 h of incubation. After an additional 24 h of incubation, the cells were harvested as previously described [5] and an aliquot removed from each sample to determine the number of cells present. The cells were th,~n frozen at --90°C tmtil the AHH assay was performed, never later tl~an 3 weeks after harvest.
315 A H H assay
Each frozen lymphocyte sample was suspended in 900/H TM buffer (pH 8) and 100/~I o f a NADPH/NADH mixture (7.5 mg o f each/ml) was added. To this reaction mixture 25 pg o f B[a]P (Sigma Chemical Company, St. Louis, MO ) in 50 ul acetone was added and all cultures were incubated for 35 rain at 37°C. Fluorometric analysis o f AHH was then performed following extraction o f phenolic BP metabolites as previously described ~I13]. Results are reported in terms o f m U o f enzyme activity where a Ur~it o f enzyme activity is defined as t h e fluorescence equiw'dent to production of 1 pmol o f 3 - h y d r o x y b e n z o [ a ] p y r e n e / m i n / m m / l O 6 cells. Values were corrected for an extmc~ion efficiency o f 60%. Values for AHH in cultured t y m p h o c y t e s were expressed as non-induced, chrysene-induced or as fold induction [(reduced lymphocyte AHH activity/non-reduced lymphocyte AHH activity} --1 ]. RESULTS AND DISCUSSION Figure 2 demonstxates the AHH induction observed in cultured, mitogens~imulated l:¢mphocytes following 24 h incubation ~dth chrysene. As shown, a s~gnificant increase in enzyme induction occurred in the chrysene-induced cultures compared witb control (non-induced) cells (1-tailed student's t-test; P < 0.001). In this particular s~t o f expeILrnents, a final concentration o f 25 #M was used to achieve induction. Although higher chrysene concentrations (50/~M) produce greater AHH ~nduction, solubility problems make higher concentrations difficult to use.
Benzonthrocene
Chrysene ~~
Benzo[cOpyrene
Fig, 2. Comparison elyAHI-Iactivity in chrysene-t~eated and control lymphocytes from 9 dif-%rent individuals. Each symbol (A--I) represents the mean o~ multiple [2--4 ] fiuorome~zlc AHH determinations performed on the same day for a ,single individual. AHH act.vity is expressed in mU of enZYmeactivity where a unit is defined .~sthe fluorescence equivalent ~o the production of } pmol of 3.hydzoxybenzo[a]pyrene/min/lO 6 cells.
316
Preliminary observations indicate ' .t chrysene-treated cellsfollow a time course of induction similarto I ~ sean with benzanthraeene or 3-MCtreated cells [14], reaching maxirnal induction approx. 24 h after incubation with the inducer. This increase in enzyme activityis linearup to 24 h, at which time a phte~u of activity is reached and maintained for the next 12 -18 h (unpublished observation ).These ~esultsare consistent with inve~,~gators w h o have observed similar cellularr~sponse~ during chrysene.stimu. lated induction i~ mammalian ceil cldtures [15]. The in vivo induction capabilitiesof chrysene have also been subsi;antiatedby studies per£ormed by Conney et al.,w h o demonstrated a 15-fc,ldincrease in placental A H H ~ollowing oral administration of chrysene (20 mg/kg) to pregnant rats [6]. In the present study, interindividualvariation in A H H inducibilityfollowing chrysene induction can be seen in Fig. 3. There is a 1.5--5.5-fold induction range obtained in this sampling. Though the sample size is small, it should be pointed out that individual variation in A H H induction by chrysene is commensurate with that seen in B A or 3-MC-induced cultured hurnan lymphoc]ftes [2]. In spite of ~he theoretical relationship between AHH induction and chemical carcinogenesis, the relationship between carcinogen metabolism and cancer susceptibility in man has not been established. Therefore, induction studies in humau tissues by compounds such as ClUTsene, might be important for llhlstrating ~,he role euzyme induction plays in the susceptibility to diseas~ caused by exogenous agents. Although chrysene per se has been shown to be metabolized by AHH to weak carcinogens only [10], the abundance of this compound in cigarette smoke might play an important role in the amplification of the AHH system by evoking increased formation of carcinogenic me~abolites of other PAHs it, the lungs of cigarette smokers. IbITERINDIVIDUAL VARIABILITY OF AHH INDUCIBILITY BY CHRYSENE
FOICl InducS~
4 3 2 I 0
A
B
C
D E F IndivldualrS
G
H
I
Fig. 3. Comparison o f mdivldual va~iatlon in lymphocyte AHH following induction by chzysene. Each symbol represents the memz of 2 --dbs ~ p l e s measured on the same day. Bars donote S.D. Fold induction is defined as {(ilJ~duced AHH activity~control A ~ activity) ~ 1 ] .
3~7 ACKNOWLEDGEMENTS The assistance of K a t h y S h u g h t e r and Vicky De Jesu,~ is g~eatly appreciated. This w o r k was s u p p o r t e d , in part~ b y grants from T he R o b e r t A. Welch F o u n d a t i o n (G-035), an NIH g r a n t (CA-15784, a gran~ from t he ACS (PDT-54), Council for Tobacco Research grants 1102 and 1094, and a grmlt f r o m the Veteran's A d m i n i s t r a t i o n Hospital, Houston, 'rexas. REFERENCES 1 Abramson, R.K. ~-~d Hutton,. J.J. (1975) Effects of cigarette smoking on AHH activity in lungs and tissues of inbred mice. Cancer Res., 35, 23--29. 2 Atlas, S.A., Vescll, B S. and l~feber~,D.W (1976) Genetic control of intenndtvidual variations in the inducibility of aryl hydrocarbon hydroxylase m cultured human lymphocytes. Cancer Res., 35, 4619---4630. 3 Badger, G.M., Donnelly, J.K. and Spotswood, T.M. (1965) The ~ormatlon of aromatic hydrocarbons at high ~,emperatures. XXIV. The pyrolysls of some tobac~ o constituents. Aust. J. Chem., 13, 1249--1266. 4 Boyum, A. (1968) lan]ation of mononuclear cells and granulocytes from human blood. Isolation of mouonuclear cel~; by one centrifugation, and of granulocytes by combimng centrifugation and .~edimentahon. Scand. J. Clin. Lab Invest., 21 (Supp. 973, 77--90 5 Busbee, D L., Shaw, C.R and Cantrel], E.T (1972) At.,,! hydrocarbon hydroxylase in human iankocytes. Science, 178, 315--316 6 Conney, A.H. (1967) Pharmacological Implications of microsomaL~enzyme ~nduction. Pharmacol. Rev., 19, 317--366. 7 Emery, A.E.H., Danford,~ H., Amand, R., Duncan, W. and Paton, L. (1978) Aryl hydrocarbon hydroxyhse inducibility in patients with cancer. Lancet, ill, 470--471. 8 Guirgis, H.A, Lynch, H.T., Mate, T., Harris, R.E., Wells, I. and Caha, L. (1976) Aryl hydrocarbon hydroxyla~e activity in lymphocytes from lung cancer and normal controls. Oncology, 33, 105--109. 9 Gurtoo, H L., Bejba, N. and Minowada, J. (1975) Properties, inducibility and an improved method of analysis of aryl hydrocarbon hydroxylase in cultured human lymphocytes. Cancer l~es, 35, 1235--1243. 10 Hecht, S.S., Bondinell, W.E. and Hoffman, D. (1974) Chrysene and methylchrysenes Presence intobaccosmokeandearcinogenic]ty.J. Natl Cancer Inst., 53, 1121--1133, 11 Kellerman, G., Cantrcll, E. and Shaw, C. (1973) Variation m extenL of aryl hydrocarbon hydroxylase induction in cultured human iymphocytes. Cancer Res., 33, .~L654~1656. 12 l~:our~ R.E.. ~tatri~, H., Atlas, S ~.., Niwa, A and Nebert, D.W (1974) Ar-fl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2~3,7,8-tetrachlorodibenzo-p-dloxin. Life Sci., 15. 1585--1595. 13 McLemore, T.L. and Martin, R.R. (1977) In vitro mduchon of aryl hydrocarbon hydroxylase m human pulmonary alveolar maarophages by benzan~hracene. Cancer Letters, 2,327--334. :/4 Nebert, D.W. and Gelboin, H.V. (1969) The m u i v o a n d m v i t r o inc~uctlon of AHH in mammalian cells of different speczes, tissues, s~trains,and developmental hormonal ~tates. Arch. Biochem. Biophys,, 134, 76--89. 15 Nebert, D.W. and Gelboin, H V. (1968) Substrate-inducible micros0mal aryl hydrocarbon hydroxylase in mammalian cell culture. II Cellular responses during enzyme reduction. J. Biol. Chem., 243, 625G~--6261. 16 Pelkoncn, D., Korhouen, P., Jouppi~a, P. and Harkl, N. (1975) Induction of ary!
318 hydrocarbon h y d r o x y ~ e in human fetal liver ,.'ells a~d fibroblast cultures by polycyclic hydrocarbons, Life Sei.~ 16, 1403--1410~. 17 Saffiotti, U.~ Moutesano, R., 8ellakumar, A.R.,, C~;fis, F. and Kaufman, D.G. (19,72) Respiratory tract carcinogenesis in hamsters induced by different numbers of administration of h enzo(a)pyren,-~ and ferrie oxide. C~r~eer Res., 32,1073--1077. 18 Welch, P.M., Loh, A and Conney, A.H. (1971) Cigarette smoke: stlmulatory effect on metabolism Qf 3,4-benzpyr~.ue by enzymes in rat lungs. Life Sci., 10, 215.
313
© KSsevier/North-HollandScientificPublishersLtd.
INDUCTION OF ARYL HYDROCARBON HYDROXYLASE IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES BY CHRYSENE
D.R. SNODGRASSI'2, T.L. McLEMORE1'3"4, M.V. MARSHALL1, N.P. WRAYs'4, E.T. CANTRELLs, D.L. BUSBEE2 and M.A. ARNOTT1 1Department of Environmental Biology, U.T M.D. Anderson Hospital a~d Tumor Institute, Houston, TX 77025, 2Department of Bmlogicol Sczences, North Texas State University, Denton, TX 76201, sZ 2partment of Pharmacology, Texas College of Osteopathze Medzcm~, Ft. Worth, TX, 3Department of Medwme, Baylor College of Medicine, Houston, TX 77025, 4Veterans Admin~strmwn Hospital, Houston, TX 77211 (U.S.A )
(Reeewed 3 May 1979) (Accepted 4 June 1979j
SUMMARY Many of the polycyclic vromatic hydrocarbons (e.g., benzo[a]pyrene (B[a] P), benzanthmcene (B A), 3-methylcholanthrene (3-MC)) are not only carcino,~enic, but also induce AHH in human tissue~. Recently, chryse~e has been implicated as an etiolo~,dc determinant of chemical carcinogenesis. Here we describe the ability of ehrysene to induce ~HH in cultured human lymphoey~es. Lymphoeytes were obtained from 9 healthy subjects, divided into 2 sets, and cultured in duplicate, triplicate, or quadruplicate for 48 h. Chrysene (25 #M final concentration) in acetone was then added to the induced[ culture set and the control set received acetone alone. Lymphocytes were then cultured ar~additkma} T24 h before harvest;ing. AHH was quantitsted by a fluorometric analysis of the phenolic metabolites produced by incubating tl~e [ymphocytes with B[a]P for 35 rain. A sigrdfieant increase in enzyme induction occurred in the chlrysene-induced cultures compared with control (non-induced) cells (one-tailed student t-test; P < 0.001). It was also observed that the interindividual vm'iation in AHH £uducibflity seen with otl~er PAHs is also observed with chrysene. IN'I~ODUCrION Aryl hydrocarbon hydroxylase ( A ~ ) is a membrane bound monooxygenase :responsible for the metabo]~ism of a wide variety of hydrophobic Address all correspondence to. Don Snodg~ss, c/o General MedicineSection (111C),
Vete~m'J~AdministrationMedical Center, 2002 Holcomb Blvd., Houston, TX 77211, U.S.A.
314 I00
1"--7Control L._J Chrysene induced
80 Achwty (milliunds) 40
A
C
D
E
F
G
H
I
lndividuols
Fig. 1. Structural comparisonbetween chlTseneand commonly used aryl hydrocarbon hydroxylase (AHH) inducers.
xenobiotics,includi~qgchemical carcinogens,to intormediate products with enhanced mutagenic and carcinogenicpotential.This enzyme complex has been implicated in the etiology of lung cancer in ,.~xparimentalanimals [1,17,18] and man [7,8,11].A H H isinduciblein human tissuesby ~arious PAHs such as benzanthmcene 3-MC, B[a]P, dimethylbenzanthmcene and tetrachlorodibenzo-p-dioxin[9,12,16]. In the present report, we have investigatedthe role of chrysene (which is similarin structureto other commonly used inducers (Fig.I) and isan environmental contaminant formed as a consequence of pyrocondensation processes,such as the combustion of tobacco products [3 ])as an inducer of A H H in cultt'Lredhuman lymphocytes. MATERIALS AND METHODS
Lymphocyte i~olation and culture techniques Venous blood (60 ml) was obtained from volunt,~ers who were not receiving medication that might he expected to alter the AHH activity of their cells. The blood was diluted 1 : i with heparinized sahne (10 units heparin/ ml saline) and mononuclear cells were separated I~.y Ficoll-Hypaque sedimentation as previously described [4]. Cells were removed from the hypaqueplasma interface and cultured by the method of McLemore et al. [13]. Two sets of cultures were incubated in either duplicate, triplicate or quadruplicate (depending on the number of cells obtained from a particular individual). One set contained cultures to which 25 #M chrysene {ICN, Irvine, CA) in 40 #l acetone had been added after 48 h of incubation. After an additional 24 h of incubation, the cells were harvested as previously described [5] and an aliquot removed from each sample to determine the number of cells present. The cells were th,~n frozen at --90°C tmtil the AHH assay was performed, never later tl~an 3 weeks after harvest.
315 A H H assay
Each frozen lymphocyte sample was suspended in 900/H TM buffer (pH 8) and 100/~I o f a NADPH/NADH mixture (7.5 mg o f each/ml) was added. To this reaction mixture 25 pg o f B[a]P (Sigma Chemical Company, St. Louis, MO ) in 50 ul acetone was added and all cultures were incubated for 35 rain at 37°C. Fluorometric analysis o f AHH was then performed following extraction o f phenolic BP metabolites as previously described ~I13]. Results are reported in terms o f m U o f enzyme activity where a Ur~it o f enzyme activity is defined as t h e fluorescence equiw'dent to production of 1 pmol o f 3 - h y d r o x y b e n z o [ a ] p y r e n e / m i n / m m / l O 6 cells. Values were corrected for an extmc~ion efficiency o f 60%. Values for AHH in cultured t y m p h o c y t e s were expressed as non-induced, chrysene-induced or as fold induction [(reduced lymphocyte AHH activity/non-reduced lymphocyte AHH activity} --1 ]. RESULTS AND DISCUSSION Figure 2 demonstxates the AHH induction observed in cultured, mitogens~imulated l:¢mphocytes following 24 h incubation ~dth chrysene. As shown, a s~gnificant increase in enzyme induction occurred in the chrysene-induced cultures compared witb control (non-induced) cells (1-tailed student's t-test; P < 0.001). In this particular s~t o f expeILrnents, a final concentration o f 25 #M was used to achieve induction. Although higher chrysene concentrations (50/~M) produce greater AHH ~nduction, solubility problems make higher concentrations difficult to use.
Benzonthrocene
Chrysene ~~
Benzo[cOpyrene
Fig, 2. Comparison elyAHI-Iactivity in chrysene-t~eated and control lymphocytes from 9 dif-%rent individuals. Each symbol (A--I) represents the mean o~ multiple [2--4 ] fiuorome~zlc AHH determinations performed on the same day for a ,single individual. AHH act.vity is expressed in mU of enZYmeactivity where a unit is defined .~sthe fluorescence equivalent ~o the production of } pmol of 3.hydzoxybenzo[a]pyrene/min/lO 6 cells.
316
Preliminary observations indicate ' .t chrysene-treated cellsfollow a time course of induction similarto I ~ sean with benzanthraeene or 3-MCtreated cells [14], reaching maxirnal induction approx. 24 h after incubation with the inducer. This increase in enzyme activityis linearup to 24 h, at which time a phte~u of activity is reached and maintained for the next 12 -18 h (unpublished observation ).These ~esultsare consistent with inve~,~gators w h o have observed similar cellularr~sponse~ during chrysene.stimu. lated induction i~ mammalian ceil cldtures [15]. The in vivo induction capabilitiesof chrysene have also been subsi;antiatedby studies per£ormed by Conney et al.,w h o demonstrated a 15-fc,ldincrease in placental A H H ~ollowing oral administration of chrysene (20 mg/kg) to pregnant rats [6]. In the present study, interindividualvariation in A H H inducibilityfollowing chrysene induction can be seen in Fig. 3. There is a 1.5--5.5-fold induction range obtained in this sampling. Though the sample size is small, it should be pointed out that individual variation in A H H induction by chrysene is commensurate with that seen in B A or 3-MC-induced cultured hurnan lymphoc]ftes [2]. In spite of ~he theoretical relationship between AHH induction and chemical carcinogenesis, the relationship between carcinogen metabolism and cancer susceptibility in man has not been established. Therefore, induction studies in humau tissues by compounds such as ClUTsene, might be important for llhlstrating ~,he role euzyme induction plays in the susceptibility to diseas~ caused by exogenous agents. Although chrysene per se has been shown to be metabolized by AHH to weak carcinogens only [10], the abundance of this compound in cigarette smoke might play an important role in the amplification of the AHH system by evoking increased formation of carcinogenic me~abolites of other PAHs it, the lungs of cigarette smokers. IbITERINDIVIDUAL VARIABILITY OF AHH INDUCIBILITY BY CHRYSENE
FOICl InducS~
4 3 2 I 0
A
B
C
D E F IndivldualrS
G
H
I
Fig. 3. Comparison o f mdivldual va~iatlon in lymphocyte AHH following induction by chzysene. Each symbol represents the memz of 2 --dbs ~ p l e s measured on the same day. Bars donote S.D. Fold induction is defined as {(ilJ~duced AHH activity~control A ~ activity) ~ 1 ] .
3~7 ACKNOWLEDGEMENTS The assistance of K a t h y S h u g h t e r and Vicky De Jesu,~ is g~eatly appreciated. This w o r k was s u p p o r t e d , in part~ b y grants from T he R o b e r t A. Welch F o u n d a t i o n (G-035), an NIH g r a n t (CA-15784, a gran~ from t he ACS (PDT-54), Council for Tobacco Research grants 1102 and 1094, and a grmlt f r o m the Veteran's A d m i n i s t r a t i o n Hospital, Houston, 'rexas. REFERENCES 1 Abramson, R.K. ~-~d Hutton,. J.J. (1975) Effects of cigarette smoking on AHH activity in lungs and tissues of inbred mice. Cancer Res., 35, 23--29. 2 Atlas, S.A., Vescll, B S. and l~feber~,D.W (1976) Genetic control of intenndtvidual variations in the inducibility of aryl hydrocarbon hydroxylase m cultured human lymphocytes. Cancer Res., 35, 4619---4630. 3 Badger, G.M., Donnelly, J.K. and Spotswood, T.M. (1965) The ~ormatlon of aromatic hydrocarbons at high ~,emperatures. XXIV. The pyrolysls of some tobac~ o constituents. Aust. J. Chem., 13, 1249--1266. 4 Boyum, A. (1968) lan]ation of mononuclear cells and granulocytes from human blood. Isolation of mouonuclear cel~; by one centrifugation, and of granulocytes by combimng centrifugation and .~edimentahon. Scand. J. Clin. Lab Invest., 21 (Supp. 973, 77--90 5 Busbee, D L., Shaw, C.R and Cantrel], E.T (1972) At.,,! hydrocarbon hydroxylase in human iankocytes. Science, 178, 315--316 6 Conney, A.H. (1967) Pharmacological Implications of microsomaL~enzyme ~nduction. Pharmacol. Rev., 19, 317--366. 7 Emery, A.E.H., Danford,~ H., Amand, R., Duncan, W. and Paton, L. (1978) Aryl hydrocarbon hydroxyhse inducibility in patients with cancer. Lancet, ill, 470--471. 8 Guirgis, H.A, Lynch, H.T., Mate, T., Harris, R.E., Wells, I. and Caha, L. (1976) Aryl hydrocarbon hydroxyla~e activity in lymphocytes from lung cancer and normal controls. Oncology, 33, 105--109. 9 Gurtoo, H L., Bejba, N. and Minowada, J. (1975) Properties, inducibility and an improved method of analysis of aryl hydrocarbon hydroxylase in cultured human lymphocytes. Cancer l~es, 35, 1235--1243. 10 Hecht, S.S., Bondinell, W.E. and Hoffman, D. (1974) Chrysene and methylchrysenes Presence intobaccosmokeandearcinogenic]ty.J. Natl Cancer Inst., 53, 1121--1133, 11 Kellerman, G., Cantrcll, E. and Shaw, C. (1973) Variation m extenL of aryl hydrocarbon hydroxylase induction in cultured human iymphocytes. Cancer Res., 33, .~L654~1656. 12 l~:our~ R.E.. ~tatri~, H., Atlas, S ~.., Niwa, A and Nebert, D.W (1974) Ar-fl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2~3,7,8-tetrachlorodibenzo-p-dloxin. Life Sci., 15. 1585--1595. 13 McLemore, T.L. and Martin, R.R. (1977) In vitro mduchon of aryl hydrocarbon hydroxylase m human pulmonary alveolar maarophages by benzan~hracene. Cancer Letters, 2,327--334. :/4 Nebert, D.W. and Gelboin, H.V. (1969) The m u i v o a n d m v i t r o inc~uctlon of AHH in mammalian cells of different speczes, tissues, s~trains,and developmental hormonal ~tates. Arch. Biochem. Biophys,, 134, 76--89. 15 Nebert, D.W. and Gelboin, H V. (1968) Substrate-inducible micros0mal aryl hydrocarbon hydroxylase in mammalian cell culture. II Cellular responses during enzyme reduction. J. Biol. Chem., 243, 625G~--6261. 16 Pelkoncn, D., Korhouen, P., Jouppi~a, P. and Harkl, N. (1975) Induction of ary!
318 hydrocarbon h y d r o x y ~ e in human fetal liver ,.'ells a~d fibroblast cultures by polycyclic hydrocarbons, Life Sei.~ 16, 1403--1410~. 17 Saffiotti, U.~ Moutesano, R., 8ellakumar, A.R.,, C~;fis, F. and Kaufman, D.G. (19,72) Respiratory tract carcinogenesis in hamsters induced by different numbers of administration of h enzo(a)pyren,-~ and ferrie oxide. C~r~eer Res., 32,1073--1077. 18 Welch, P.M., Loh, A and Conney, A.H. (1971) Cigarette smoke: stlmulatory effect on metabolism Qf 3,4-benzpyr~.ue by enzymes in rat lungs. Life Sci., 10, 215.