Naunyn-Schmiedeberg's Arch Pharmacol (1993) 348:435-443

Naunyn-Sch miedeberg's

Archivesof Pharmacology © Springer-Verlag 1993

Response of mouse liver coumarin 7-hydroxylase activity to hepatotoxins: dependence on strain and agent and comparison to other monooxygenases

Pertti Pellinen 1, Frej Stenb~ck 2, Arj a Rautio 3, Olavi Pelkonen 3, Matti Lang l . , and Markku Pasanen 1,3

1Department of Pharmacology and Toxicology, University of Kuopio, PL 1627, SF-70211 Kuopio, Finland 2Department of Pathology, University of Oulu, SF-90220 Oulu, Finland 3 Department of Pharmacology and Toxicology, University of Oulu, SF-90220 Oulu, Finland

Received October 6, 1992/Accepted June 25, 1993

Summary. Acute effects of a single intraperitoneal dose of allyl alcohol (AA, 64 mg/kg), dimethylnitrosamine (DMNA, 30 mg/kg), hexachlorobutadiene (HCBD, 50 mg/kg), carbon tetrachloride (CC14, 24 mg/kg), co- caine (60 mg/kg) and pyrazole (300 mg/kg) on the hepat- ic histology and monooxygenases in DBA/2 and C57B1/6 strains of mice were investigated. All substances caused histologically verified injury to the liver, which varied in appearance and severity depending on the compound and the mouse strain. Responses of P450-catalyzed reactions were highly dependent on the toxin and varied between different monooxygenase (MO) reactions and two mouse strains. In DBA/2 strain, coumarin 7-hydroxylase (COH) activity was increased from 3- to 5-fold by pyrazole, co- caine, HCBD and CC14. With respect to P450 content and other MO activities, no changes or even decreases were generally observed. Some exceptions to this rule were found: HCBD significantly increased T15aOH, PROD and EROD activities in C57B1/6 mice, whereas cocaine caused a significant stimulation of T l5a OH and PROD in DBA/2 mice, It is concluded that i) different hepatoxins cause different types of liver injury and responses of the monooxygenase complex ("hepatotoxin- specific finger prints"), ii) although DBA/2 and C57B1/6 mice responded rather similarly to hepatotoxins, also with respect to P450 content and most MO activities, they

Abbreviations: COH, coumarin 7-hydroxylase; P450Coh, a P450 isozyme active in coumarin 7-hydroxylation; T 15 a OH, testosterone 15 a-hydroxylase; P 450 T 15 a OH, a P 450 enzymes catalyzing testos- terone 15a-hydroxylation; EROD, 7-ethoxyresorufin O-deethylase; PROD, 7-pentoxyresorufin O-dealkylase; AHH, aryl hydrocarbon hydroxylase; MO, monooxygenase; SDS, sodium dodecyl sulphate; DMNA, dimethylnitrosamine; CC14, carbon tetrachloride; HCBD, hexachloro-l,3-butadiene; AA, allyl alcohol

An update list and suggested nomenclature of the sequenced P450 genes has been published recently (Nelson et al., DNA Cell Biol 12:1-51, 1993). The suggested nomenclature is followed in this manuscript. The murine P450Coh is the product of the Cyp2a-5 gene (the human counterpart is CYP2A6) and the murine P 450T 15 a OH is the product of the Cyp2a-4 gene

Correspondence to: M. Pasanen at the above address

displayed a profound difference in the behaviour of COH activity, and iii) within the P450 superfamily, the regula- tion of COH activity seems to be rather unique, also when compared to its structurally close enzyme, testosterone 15a-hydroxylase.

Key words: Coumarin - P450Coh - Drug metabolism - Hepatotoxicity - Liver damage

Introduction

Coumarin, a plant alkaloid and a substrate for xenobiotic-metabolizing P450 reactions (Cohen 1979; Egan et al. 1990), is 7-hydroxylated by a specific P450Coh isozyme coded by Cyp2a-5 gene in mouse and CYP2A6 in man (Negishi et al. 1989; Lang et al. 1989; Yamano et al. 1990). The regulation of this enzyme dis- plays some unique characteristics. In addition to pheno- barbital (Wood and Conney 1974; Juvonen et al. 1985), many other structurally unrelated compounds are able to increase the activity of COH in mouse liver. These in- clude e.g. TCPOBOP (Raunio et al. 1988; Honkakoski et al. 1992), pyrazole and its derivatives (Juvonen et al. 1987; Kojo et al. 1991), pyrazine (Hahnemann et al. 1989), cobalt (Legrum and Netter 1980; Kocer et al. 1991), indium (Mangoura et al. 1989) and cerium (Arvela et al. 1991). Some of these compounds are hepatotoxic and decrease the total P450 content as well as several monooxygenase activities catalyzed by various P450 en- zymes (Juvonen et al. 1985, 1987; Arvela et al. 1992). Therefore, it seems that within the P450 superfamily, the COH activity may be exceptional in its response to hepatotoxins.

Most of hepatic cytochrome P450 content is concen- trated in perivenous hepatocytes (Kanai et al. 1986) with a slightly increasing periportal-perivenous gradient (Kanai et al. 1990; Seibert et al. 1989). Therefore, enzyme activities, such as aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxy- resorufin O-deethylase (EROD), benzphetamine N-deme-

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thylase and mic rosoma l e thano l oxida t ion , represent ing P450 families 1A, 2B, 2 E and 3A, are main ly local ized to the per ivenous zone (Baron et al. 1986; Vfi~in~inen 1986; Bengtsson et al. 1987; Seiber t et al. 1989; Gascon- Barre et al. 1989). However, the loca t ion o f C O H under chemical stress is still unknown.

Some wel l -known hepa to tox ins such as CCI 4 and co- caine have been repor ted to decrease the to ta l P450 con- tent (Okino et al. 1991) and cer ta in hepa t ic xenobio t ic - me tabo l i z ing activi t ies (Marsha l l and McLean 1969). The except ional response o f C O H accompany ing the liver h is to logica l pa ramete r s in liver in jury is unknown. There- fore this i n f o r m a t i o n would a id in unde r s t and ing the role o f P4502a-5 in liver in jury and poss ib ly in preventing tox- icological effects o f chemicals in man . We sought to ex- tend our studies to some well k n o w n yet d i f ferent ly ac t ing hepato toxins . To this a im two inbred strains o f mice, D B A / 2 and C57BL/6 , different in their C O H inducibi l i - ty ( Juvonen et al. 1985, 1987), were t rea ted with pyrazole, cocaine, d imethy ln i t rosamine , c a rbon te t rachlor ide , hexa- ch lo robu tad iene and al lyl a lcoho l and their acute effects on C O H were ana lyzed and c o m p a r e d to o ther m o n o - oxygenases representat ive o f different P450 enzymes. His to log ica l evaluat ions and P450 form-speci f ic enzyme assays were p e r f o r m e d to s tudy the poss ib le l inkages be- tween liver in jury and changes in the P450 profi le.

Materials and methods

Chemicals: Coumarin, 7-hydroxycoumarin, 7-ethoxy- and 7-pentoxy- resorufin, benzo(a)pyrene, pyrazole, DMNA, Tween 80, bicinchonic ac- id, NADPH, 5-bromo-4-chloro-3-indolyl phosphate, and goat anti-rab- bit IgG alkaline phosphatase were purchased from Sigma (St. Louis, MO, USA). Allyl alcohol, CC14, and silica gel G 60 F 254 plates were from E. Merck (Darmstadt, Germany). Hexachloro-l,3-butadiene (HCBD) was obtained from Aldrich Co. (Milwaukee; WI, USA). Co- caine hydrochloride was from Yliopiston Apteekki (Helsinki, Finland). 4-(14C)testosterone was bought from Amersham (Bucks, UK). Nitrocellulose sheets were from Schleicher & Schuell (Dassel, Germa- ny). Electrophoresis reagents were purchased from Bio-Rad (Richmond, CA, USA). All other chemicals were of at least analytical grade.

Treatment of animals. Male DBA/2/KUO (D2) and C57BL/6N/KUO (B6) mice, 7 - 10 weeks old, were obtained from the National Laborato- ry Animal Center at the University of Kuopio. They were housed in macrolon cages in groups of 5-10 mice under standard laboratory conditions: 12/12 h light/dark c y c l e (7:00 am- 19.00 pm/ 19:00 pro-7:00 am), 22_+ 1 °C temperature and 50-60°/0 humidity. Standard rodent feed (Ewos, Sweden) and tap water were available ad libitum. All chemicals were given to the animals (4-6/group; 20-24 animals at a time; weight 20-25 g) intraperitoneally dissolved in saline or corn oil in a volume of 0.1 ml/10 g body weight at 8 -9 o'clock a.m. 24 h before sacrifice. Treatments were as follows: DMNA 30 mg/kg, pyrazole 300mg/kg, CC14 24mg/kg, cocaine 60mg/kg, HCBD 50 mg/kg, and AA 64 mg/kg. The dose selection was based on the data in the literature where unequivocal liver damage was obtained. The con- trol animals were dosed with the same volume of saline or corn oil. All animals were fasted overnight before sacrifice.

Histopathology and preparation of microsomes. At autopsy the liver was removed, and a representative sample was fixed in 10°70 neutral buf- fered formalin. After fixation, the samples were processed for histopathological examination. Serial sections, 5 ~tm thick, were stained with hematoxylin and eosin and other stains where needed. The remain- der of the liver was used for the preparation of microsomes (Lang et al. 1989).

Determination of monooxygenase activities. Microsomal protein con- centrations were determined by the bicinchonic acid method (Smith et al. 1985); cytochrome P450 content according to the method of Omura and Sato (1964); the COH activity was measured by the method of Aitio (1978) using 100 ~M coumarin as a substrate instead of 7-ethoxy- coumarin; EROD and PROD activities using the end-point method of Burke et al. (1977); the AHH activity according to Nebert and Gelboin (1968), and T15aOH activity according to Waxman et ai. (1983).

Western blotting. The preparation and validation of anti-P450Coh anti- body has been reported by Lang et al. (1989). It recognizes both P450Coh and a structurally close enzyme P450T15a OH (Raunio et al. 1988). Microsomal proteins (15-20 ~g) were separated by electrophore- sis on 9°70 SDS-polyacrylamide gel according to Laemmli (1970). After transferring the proteins to nitrocellulose sheets (Towbin et al. 1979), processing was continued according to modifications described earlier (Honkakoski and Lang 1989; Honkakoski et al. 1992). Antibody-pro- tein complex was visualized with 5-bromo-4-chloro-3-indolyl phos- phate, and the intensitiy of the bands was quantitated with a Shimadzu CS-9000 dual wavelength scanner.

Statistical analysis. The values of enzyme activities are expressed as means (+ S.D., n = 4-6) and statistical significance was determined by Mann-Whitney U-test. P-values less than 0.05 were considered statisti- cally significant.

Results

Histological examination

Since the asce r ta inment o f liver in jury was a bas ic prereq- uisite for the goals o f the study, we evaluated the mor- pho log ica l appea rance o f livers af ter different t rea tments . Dis t inct a l te ra t ion o f the n o r m a l s t ructure in all groups was found (Figs. 1, 2). Liver changes consis ted o f the swelling o f hepatocytes , i r regular i ty in the size and shape o f cells and nuclei, and a h o m o g e n o u s ac idophi l ic stain- ing o f cytoplasm. I n f l a m m a t i o n , f ibrosis and hemor rhag - es were u n c o m m o n as the specimens were examined 24 h af ter insult . Cel lu lar necrosis was observed in the an imals exposed to CC14 (severe) and, to a lesser extent in case o f D M N A , and in one case o f A A (Figs. 1, 2). Coca ine and H C B D t rea tments p r o d u c e d a var iable degree o f diffuse liver in jury in the area su r round ing the central vein. Af te r pyrazole t r ea tment the d a m a g e was loca ted in the area su r rounding the central vein and in the in te rmedia ted ar- ea. D M N A - i n d u c e d in jury was loca ted in the cen- t r i lobu la r area. CC14 p roduced per ivenous in jury and A A - i n d u c e d in jury was seen in the pe r ipo r t a l cells. CC14 induced liver damage was more severe in D B A / 2 than in C57B1/6 mice, while the reverse effect was found for H C B D and D M N A . The f indings are summar ized in Fig. 3.

Enzymat ic analysis

Percentage changes, toge ther with s ta t is t ical s ignif icance o f the differences, in to ta l cy tochrome P450 content and the C O H act ivi ty and o ther M O s measured are presented in Fig. 4.

Coumarin 7-hydroxylase

C o m p a r e d to o ther MOs, the response o f the C O H activi- ty was s t r ik ingly different. A s ignif icant f rom 3- to 5-fold increase in the C O H act ivi ty t o o k place, pa r t i cu la r ly in

437

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Fig. 1. Liver structure in C57B1/6N mice exposed to allylacohol (A), CC14 (B), HCBD (C), pyrazole (D), cocaine (E) and DMNA (F) show- ing varying degrees of liver injury: cell necrosis, cytoplasmic homo-

geneity and nuclear enlargement (H-E, original magnification 240x, * = central vein hepatocyte, ~ shows the area of necrosis)

the DBA/2 mice, after pyrazole, HCBD, CC14 and co- caine treatments (Fig. 4). This increase was accompanied by an increase in the amount of microsomal P450Coh as detected in Western blots (Fig. 3). However, in micro- somes from CC14-treated mice the amount of P450Coh did not parallel with the increased COH activity: the amout of the immunolabelled protein did not increase when compared to the corresponding control value. In C57BL/6 mice, only AA and pyrazole caused a less than 2-fold increase in the COH activity, whereas other hepatotoxins were either without effect (HCBD, CC14,

cocaine) or caused a decrease (DMNA) in this activity (Fig. 4).

Testosterone 15a-hydroxylase

Because this enzyme is structurally very similar to P450Coh, it was of interest to measure its response to dif- ferent hepatotoxins. DMNA decreased the activity by about 50°7o in both strains, and less than 2-fold increases were observed after HCBD (C57BL/6) and cocaine (DBA/2) pretreatments. Corn oil as a vehicle decreased

438

Fig. 2. Liver structure in DBA/2 mice exposed to allyl alcohol (.4.), CC14 (B), HCBD (C), pyrazole (D), cocaine (E) and DMNA (17) show- ing varying degrees of fiver injury: alteration in liver cell and nuclear

size and shape mostly around the central vein (*) hepatocyte, -~ shows the area of necrosis (H-E, magnification 240×)

COH by 30°70 and T 1 5 a O H by 20°70 in both strains of mice.

and HCBD in C57BL/6 and by cocaine in DBA/2 mice; EROD was increased by HCBD (Fig. 4).

Other MOs

Four other MO activities catalyzed by different P450 isozymes and the total P450 content were also deter- mined. In most cases, particularly in the case of the total P450 content and the A H H activity, the hepatotoxins ei- ther significantly decreased or did not change these pa- rameters (Fig. 4). Some exceptions to this rule were ob- served: PROD was significantly increased by pyrazole

Strain differences

COH activity seems to be unique amongst the MOs also with respect to its strain dependent responses to hepatotoxins. The strong stimulatory effects could main- ly be seen in the DBA/2 mice (Fig. 4); the only exception was AA causing a slightly stronger increase in C O H acitivity in the C57B1/6 than DBA/2 mice. HCBD exhib- its a stronger stimulatory effect on EROD and T15a OH

439

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Fig. 3. Severity of liver injury and immunodetection of P4502a-4/5. (A) The histogram shows a semiquantitative strain-dependent difference in liver injury between DBA/2 and C57B1/6 mice, I = slight; 2 = moder- ate; 3 = severe; + = more severe than in the corresponding strain. Allyl alcohol (AA), carbon tetrachloride (CC14), hexachlorobutadiene (HCBD), pyrazole (PYR), cocaine (Coc), dimethylnitrosamine (DMNA). (B) The histogram demonstrates fold increases in the amount of P4502a-4/5 protein. On the basis of COH activity determinations only those samples with significant changes were used in immunoblot- ting experiments. Four individual determinations in DBA/2 (]:)2) mouse liver microsomes were carried out; one representative sample was ana- lyzed further in C57B1/6 (B6) mouse liver microsomes. Specific P4502a-4/5 contents in D2 microsomes were as follows (pmol/mg pro- tein; mean+s.d.): Control: 31+12; PYR: 147+39. Control: 11+3; HCBD: 24_+2; CC14: 15_+6. Control: 20_+5; Coc: 97_+31

in C57B1/6 than DBA/2. On the other hand, CCI 4 de- creased the total P450 content, and EROD and PROD ac- tivities more in DBA/2 than in C57B1/6. Cocaine is par- ticularly interesting because it stimulates T15aOH and PROD in the DBA/2 mice and decreases PROD in the C57B1/6 mice (Fig. 4).

Discussion

The present study describes the acute effects of six struc- turally different hepatotoxins on P450-catalyzed mono- oxygenase enzymes in association with the histologically characterized liver injury. The MO activities were selected to represent some well known xenobiotic metabolizing P450 enzymes and corresponding individual genes or gene families in the mouse liver: EROD is closely linked to l a (mainly la-1), PROD is a marker for 2b, COH for 2a-5, T 1 5 a O H for 2a-4 and A H H for l a and 2c (for a review see Gonzalez 1988). Particularly we were interested in the COH activity, catalyzed by Cyp2 a-5 gene preduct P450Coh, which is readily increased in the mouse liver af- ter various pretreatments (see the Introduction) and which was shown here to be increased several-fold by the 24-hour treatments by pyrazole, HCBD, cocaine, AA and CCI 4.

In terms of xenobiotic metabolism, the two mouse strains differ remarkably from each other. The COH ac- tivity and inducibility is controlled by the Coh locus (Wood and Conney 1974; Lush and Arnold 1975; Wood 1979), DBA/2 being the responsive strain to pyrazole and phenobarbital (Juvonen et al. 1985, 1987, Lang et al. 1988). In the present study we demonstrated that a similar differential responsiveness between the two strains generally applies to the hepatotoxins studied here: HCBD, CCI 4 and cocaine increased the COH activity only in DBA/2 mice, whereas AA and pyrazole were ef- fective also in C57BL/6 mice.

AHH and EROD activities are linked to the Ah locus in mice. They are inducible by polycyclic aromatic hydro- carbons like 3-methylcholanthrene in C57B1/6 mice while DBA/2 mice are nonresponsive (Nebert et al. 1975, 1982). COH activity and inducibility are not linked in any way with this locus, as confirmed also by the present results.

The P4502a-5 catalyzing COH activity has a 98.3°7o homology with Cyp2a-4 responsible for T15aOH activi- ty. They differ from each other in only 11 amino acid resi- dues and immunologically they are identical (Negishi et al. 1989; Lindberg et al. 1989). The regulation of Cyp2a-4/5 may be strain dependent. Phenobarbital in- creases the transcription rate of Cyp2a-4/5 in C57B1/6 but not in DBA/2 mice while pyrazole and cobalt in- crease the COH activity by postranscriptional mecha- nisms in C57B1/6 mice (Aida and Negishi 1991; Hahne- mann et al. 1992). The present results demonstrate that the T15aOH activity did not parallel that of COH with any treatment, except cocaine, indicating that the two closely related P450 isozymes are differently regulated, not only in liver and kidney (Squires and Negishi 1988), but also in their response to a number of hepatotoxic agents.

Our basic hypothesis in the beginning of the study was that the increase in COH activity is associated with the acute liver damage. In general, inducers of xenobiotic metabolizing enzymes will change the distribution profile of P450s, mainly in a chemical-dependent manner (Gebhardt 1992). Cocaine, AA, HCBD, CCI 4 and pyrazole act as inducers and are hepatotoxic. In fact, these five chemicals increased P4502a-5 and associated COH activity three to five fold compared with control levels while total cytochrome P450 content was not affect- ed or rather decreased.

The localization of liver injury and occurrence of catalytical "active" cells and the amount of COH activity was studied. However, because in this work no immuno- histochemistry was done the exact enzyme specific zona- tion cannot be made. Pyrazole is a potent inducer of COH and produces a generalized liver injury except a narrow cell border around the central vein. Therefore, it is unlikely that the remaining (increased COH) activity should locate only in healthy perivenous cells of a narrow zone around the central vein. Cocaine- and HCBD-in- duced liver injury is located around the central vein and the enhanced activity could be located in the periportal cells. CC14 produced a severe injury around the central vein, AA causes hepatic injury also within the same cells and the remaining activity should locate only in peripor-

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Fig. 4. Effect of hepatotoxic agents on mouse hepatic microsomal cytochrome P450 content and monooxygenase activities in male DBA/2 (D2) and C57BL/6 (B6) mice. Coumarin 7-hydroxylase (COH), testos- terone 15a-hydroxylase (T15aOH), 7-pentoxyresorufin O-dealkylase (PROD), 7-ethoxyresorufin O-deethylase (EROD) and aryl hydrocar- bon hydroxylase (AHH). Doses were: dimethylnitrosamine (DMNA), 30 mg/kg body weight; pyrazole (Pyr), 300 mg/kg; allyl alcohol (AA), 64 mg/kg; hexachloro-l,3-butadiene (HCBD), 50 mg/kg; carbon tetrachloride (CC14), 24 mg/kg; mg/kg; cocaine (Coc), 60 mg/kg. All chemicals were administered as a single i.p. injection 24 h before sacrify- ing the animals. P450 content (nmol/mg protein) and enzyme activities

(pmol/min/mg protein) are expressed as percentage of control activity. Each bar represents mean-+SD of 4 - 6 animals, *=p<0 .05 , ** = p < 0.01. Control values of P450 content were 0.492 + 0.080 nmol/ mg and 0.542+0.088 nmol/mg microsomal protein in D2 and B6, re- spectively. The ranges of COH activity values in control D2 and B6 mice were as follows: among saline controls 37.6_+ 11.0 pmol/mg/min and 34_+14.1 pmol/mg/min and corn oil controls 9_+2 pmol/mg/min and 20 + 6 pmol/mg/min, respectively. The range in control values of other monooxygenases in D2 and B6 mice was as follows (pmot/mg/min): T15aOH, 155-200 and 150-236; PROD, 21-40 and 14-28; EROD, 616-1514 and 988-1782; AHH, 100-131 and 86-116, respectively

tal cells. These results show that the location of injury varies greatly depending on the injuring chemical, and it is hard to say that the "remaining" enhanced COH activi- ty is only located in "health" cells. This conclusion sup- ports earlier studies which demonstrated that also some other hepatotoxins, notably cobalt (Legrum and Netter 1980; Kocer et al. 1991) and cerium (Arvela et al. 1991; Salonp~i~ et al. 1992) increase COH activity several-fold despite overt liver injury.

At the dose used DMNA caused liver necrosis. Simul- taneously, both the P450 content and all the MOs tested decreased. Against our working hypothesis only DMNA did not increase the COH activity in either strain despite causing over liver damage. A possible explanation may be that a relatively high dose was used in this experiment. It has been reported recently that a low dose of nitrosoamines is already enough to cause DNA damage (Brambilla et al. 1992). Hence, higher doses may be ex-

ceedingly toxic for cells. However, an experiment using lower doses (1 g g - 1 mg/kg) did not induce C O H activity (results not shown). Another possibility is that D M N A serves as a "suicide substrate" for COH resulting in decreased enzymatic activity. In relation to this a recent study (Camus et al. unpublished) shows that indeed DMNA is a good substrate for P450Coh in mouse and man.

In rat liver AA produces periportal necrosis (James et al. 1981; Greus et al. 1982) and decreases in cytochrome P450 content as well as several associated monooxygenase activities (Thorgeirsson et al. 1976; Greus et al. 1982). In the present study, necrosis was observed in both strains, but significant changes, a slight decrease of both total P450 content and A H H activity and an increase in COH, were found only in C57BL/6 mice which in standard con- ditions according to the Coh locus studies is C O H "nonresponsive". Again, a low dose (1 ~tg-1 mg/kg) experiment did not induce COH activity (results not shown).

CC14 has been documented to decrease hepatic microsomal P450 content as one of the earliest response of rat liver (Smucler et al. 1967; Greus et al. 1982) occur- ring simultaneously with the production of the highly re- active metabolite, trichloromethyl radical (Nogushi et al. 1982). When centrilobular liver cells are damaged both phenobarbital-inducible proteins together with several enzymatic activities e.g. aminopyrine N-demethylase, benzo(a)pyrene hydroxylase, 7-ethoxycoumarin O-de- ethylase and polycyclic aromatic hydrocarbon-inducible activities e.g. 7-ethoxyresorufin O-deethylase will de- crease (Head et al. 1981; Moddy et al. 1986; Letteron et al. 1990; Sohn et al. 1991; Greus et al. 1982). Therefore, our results are in line with the earlier studies. Here, ac- cording to the assumption, the most severe necrosis was detected in COH responsive DBA/2 strain, and unlike the other MOs, either phenobarbital-inducible or the ac- tivities controlled by the Ah locus, COH activity in- creased in DBA/2 mice, although in protein level this in- crease was not equally clear.

It has been shown that a single injection of cocaine produces hepatotoxicity in mice in a dose-and time-de- pendent manner (Kloss et al. 1982). Here, cocaine as well as HCBD treatment produced a diffuse necrosis around the central vein of same degree in both strains of mice but only DBA/2 strain responded with increases in COH ac- tivity and corresponding protein.

These results strengthen the concept that liver damag- ing agents do not evenly depress xenobiotic metabolizing activities. Consequently, one may hypothesize that differ- ent hepatotoxic agents may display unique "finger- prints" with respect to P450 enzymes. In this respect the COH activity behave rather uniquely amongst P450 isozymes as examplified by CC14 administration. Espe- cially interesting was to find that cocaine enhanced all the activities tested to a different extent despite the fact that it caused a clear liver injury. We are currently studying co- caine-induced injury more extensively.

Corn oil as a vehicle for the control animals decreased only COH and T 1 5 a O H activities in the both strains of mice, having no effect on the total P450 content or the

441

other xenobiotic metabolizing activities tested, and still, no liver damage was observed (results not shown). This finding agrees with another study (Honkakoski et al. 1992), where a similar decrease in COH activity was ob- served among olive oil treated control animals. On the other hand, this vehicle did not suppress the C O H induc- tion. At the moment we have no plausible explanation for this phenomenon.

Our present results demonstrate that the two strains of mice (DBA/2 and C57BL/6) differ greatly from each other with respect to their COH response. DBA/2 strain exhibits a large increase in COH activity after pyrazole, HCBD, CC14 or cocaine, while in the C57B1/6 strain COH activity remains at the control level. Moreover, total cytochrome P450 levels were not conspicuously different and the two strains did not exhibit any remarkable strain- dependent differences in terms of other P450 reactions ei- ther linked to phenobarbital-type induction or controlled by the Ah locus.

Attempts to associate the type of injury and the re- sponse of COH yielded inconclusive results. One possible reason may be that the chemicals used in this study have different mechanisms and potency by which they cause liver damage. Potentially, a low COH activity could mean a high toxic potency of the chemical. D M N A and AA did not induce C O H in a high or low dose. Both of them de- creased total P450 content and the other MOs tested. However, it may be of interest to note that the increase in COH activity is associated also with neoplastic lesions in the mouse liver (Lange et al. 1990). Is the increase in COH activity a signal for or just coincidental to an acute liver damage, or could it be an early signal for ensuing chronic cell alterations, such as cancer? We have no defi- nite answer at present. Nevertheless, whatever is the mechanism behind the exceptional expression of P4502a-5 protein and associated COH activity, it is dif- ferent from the other P450s.

Acknowledgements. We thank Ms P/~ivi Kylli, Ms Mirja Vahera, Ms Er- ja Tomperi and Ms Riitta Heikkinen for their skillful technical assis- tance and Drs Hannu Raunio and Paavo Honkakoski for their contribu- tion. This work was supported financially by the Academy of Finland, Medical Council contract no: 1051029 and the Project Grants of the University of Kuopio.

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