Arch Toxicol(1992) 60:560-566
Archives of
Toxicology 9 Springer-Verlag1992
Distribution and inducibility of a P450I activity in cellular components of the avian immune system Nancy A. Lorrl, 3, Karen A. Golemboski% 3, Richelle A. Hemendinger], Rodney R. Dietert 2, 3, and Stephen E. Bloom1, 3
1Departmentof Avianand AquaticAnimalMedicine, 2 Departmentof Microbiology,Immunology,and Parasitology,and J Institutefor Comparative and EnvironmentalToxicology,ComellUniversity,Ithaca,New York,USA Received 20 January 1992/Accepted18 May 1992
Abstract. The level of expression of the cytochrome P450 system in an immune tissue could influence the sensitivity of that irmnune tissue to damage by xenobiotics. The capacity of immune organs and their cellular components for P450I-catalyzed metabolism was assayed in the 4-weekold chicken using the P450I-specific ethoxyresorufin-Odeethylase (EROD) assay and the P450I-inducer, 3,4,3',4'tetrachlorobiphenyl (TCB). After induction by TCB, EROD was detectable in microsomes from whole thymus, bursa and in peritoneal exudate cells (containing primarily macrophages) at levels of 28.3, 7.2 and 1.3 pmol/mg microsomal protein/min, respectively; the level in control liver was 89.9 pmol/mg microsomal protein/min. No activity was detected in these immune tissues without induction. The P450I specific in vitro inhibitor, ct-naphthoflavone (NF) inhibited the TCB-induced liver and immune tissue EROD by 50% at concentrations in the range of 0.07-0.1 jaM. The cellular distribution of EROD in the bursa and thymus was studied in lymphocytes and supporting tissue cells after their separation by density gradient centrifugation. Much higher TCB-induced EROD was detected in immune tissue supporting cells than in lymphocytes, particularly in the thymus. The P450I in the supporting tissue of the bursa and thymus at 1 week posthatch was also measured after eradication of the lymphocytes in both immune tissues by in ovo administration of CP. TCB-induced EROD was 12-fold higher in the lymphocyte-depleted thymus than in normal thymus, with a less marked but similar pattern in the bursa. These studies suggest that after exposure to a P450I inducer, levels of CP, cyclophosphamide;TCDD, 2,3,7,8-tetrachlorodibenzodioxin; TCB, 3,4,3",4'-tetrachlorobiphenyl;EROD, ethoxyresorufinO-deethylase; NF, ct-naphthoflavone;PB, sodium phenobarbital; BCA, bicinchoninicacid; PECs, peritoneal exudate cells; i.p., intraperitoneal; b.w., body weight;PBS, phosphatebufferedsaline; DI, days of incubation; WPH, weeks posthatch; DPH, days posthatch; BZOD, benzyloxyphenoxazone-o-debenzylase; ICs0, 50% inhibitory concentration; DMSO, dimethylsulfoxide;BE benzo(a)pyrene
Abbreviations:
C o r r e s p o n d e n c e to: Nancy A. Lorr, Dept. Avian & Aquatic Animal Medicine, CornellUniversity,203 Rice Hall, Ithaca, NY 14853, USA
P450I are much higher in the supporting cells of immune organs than in the immune cells themselves.
Key words: P450I
Chicken
-
Immune tissue - Cytochrome
Introduction
I I
The sensitivity of an immune tissue to xenobiotic-induced damage may depend on the capacity of that immune tissue for metabolic activation of a particular toxin to forms which react with DNA and other macromolecules. The hepatic cytochrome P450 monooxygenase system oxidizes xenobiotics and endogenous substrates, transforming some molecules to reactive electrophilic intermediates. While some electrophilic intermediates are sufficiently stable to be transported from the liver to a target organ, or are carded from the liver in a transport form, the high reactiv-! ity and instability of some P450 metabolites may preclude translocation to the target organ. In the latter cases, target organ P450 may be responsible for toxicity. P450IA1 is induced by polycyclic aromatic hydrocarbons (e. g. 3-methylcholanthrene) and coplanar chlorinated hydrocarbons (e. g. 2,3,7,8-tetrachlorodibenzodioxin (TCDD)), and it also metabolizes most of these inducers. Basal and induced P450IA1, at low levels, has been detected in a variety of immune tissues including cultured human lymphocytes (Whitlock et al. 1972; Amsbaugh et al. 1986), macrophages from several species (Peters0n 1987), murine spleen (Blank et al. 1987; Kawabata and White, Jr. 1989) and rabbit bone marrow (Schnier et al. 1989). P450I-catalyzed activity was detected in the chicken thymus (Nikolaidis et al. 1988b; Misra et al. 1991) and bursa (Misra et al. 1991) after pretreatment with P4501 inducers. The P450I(s) form in chicken liver is similar to both P450IA1 and P450IA2 in mammals in its catalytic activity and inducibility (Hokama et al. 1988; Jacobs et a/.
561 Table 1. EROD and BZOD in microsomes from liver and lymphoid organs from control and induced 4 week old chickens. 1 EROD and BZOD is in prnol/mgmicrosomal protein/min. Values are means + standard deviations; the numbers in parentheses are the numbers of samples Organ2
Treatment
EROD
Liver
Corn Oil TCB Water PB
89.9 _+ 19.0 6363.3 • 1264.6 139.6 • 1.5 61.7 ___ 4.2
Bursa
Corn oil TCB
0.0 7.2 •
2.5
(5) (5)e
0.0 1.8 ...
Corn oil TCB
0.0 28.3 ___
9.5
(5) (5)b
0.0 4.6
(1) (1)
Corn oil TCB
0.0 1.3 ___
0.9
(4) (4)e
0.0 0.0
(1) (1)
Thymus PECs
BZOD (6) (12)a* (2) (2)
26.4 _ 6.2 1297.8 "+"254.8 33.7 _+ 0.3 20.5 + 0.3 0.6
(4) (3) (2) (2) (2) (2)
I Assaysrun with I p.M ethoxyresorufin and 5 I.tM benzyloxyphenoxazone 2 Microsomal preparations of liver, bursa, and thymus were made directlyfrom whole organ sonicated homogenates. Microsomal preparationsfrom PECs were made from washed and sonicated peritoneal exu-
date cells. All samples were stored frozen at -80 ~C * In comparing TCB-induced EROD of microsomes from different tissues, ERODs are statistically different (p
Archives of
Toxicology 9 Springer-Verlag1992
Distribution and inducibility of a P450I activity in cellular components of the avian immune system Nancy A. Lorrl, 3, Karen A. Golemboski% 3, Richelle A. Hemendinger], Rodney R. Dietert 2, 3, and Stephen E. Bloom1, 3
1Departmentof Avianand AquaticAnimalMedicine, 2 Departmentof Microbiology,Immunology,and Parasitology,and J Institutefor Comparative and EnvironmentalToxicology,ComellUniversity,Ithaca,New York,USA Received 20 January 1992/Accepted18 May 1992
Abstract. The level of expression of the cytochrome P450 system in an immune tissue could influence the sensitivity of that irmnune tissue to damage by xenobiotics. The capacity of immune organs and their cellular components for P450I-catalyzed metabolism was assayed in the 4-weekold chicken using the P450I-specific ethoxyresorufin-Odeethylase (EROD) assay and the P450I-inducer, 3,4,3',4'tetrachlorobiphenyl (TCB). After induction by TCB, EROD was detectable in microsomes from whole thymus, bursa and in peritoneal exudate cells (containing primarily macrophages) at levels of 28.3, 7.2 and 1.3 pmol/mg microsomal protein/min, respectively; the level in control liver was 89.9 pmol/mg microsomal protein/min. No activity was detected in these immune tissues without induction. The P450I specific in vitro inhibitor, ct-naphthoflavone (NF) inhibited the TCB-induced liver and immune tissue EROD by 50% at concentrations in the range of 0.07-0.1 jaM. The cellular distribution of EROD in the bursa and thymus was studied in lymphocytes and supporting tissue cells after their separation by density gradient centrifugation. Much higher TCB-induced EROD was detected in immune tissue supporting cells than in lymphocytes, particularly in the thymus. The P450I in the supporting tissue of the bursa and thymus at 1 week posthatch was also measured after eradication of the lymphocytes in both immune tissues by in ovo administration of CP. TCB-induced EROD was 12-fold higher in the lymphocyte-depleted thymus than in normal thymus, with a less marked but similar pattern in the bursa. These studies suggest that after exposure to a P450I inducer, levels of CP, cyclophosphamide;TCDD, 2,3,7,8-tetrachlorodibenzodioxin; TCB, 3,4,3",4'-tetrachlorobiphenyl;EROD, ethoxyresorufinO-deethylase; NF, ct-naphthoflavone;PB, sodium phenobarbital; BCA, bicinchoninicacid; PECs, peritoneal exudate cells; i.p., intraperitoneal; b.w., body weight;PBS, phosphatebufferedsaline; DI, days of incubation; WPH, weeks posthatch; DPH, days posthatch; BZOD, benzyloxyphenoxazone-o-debenzylase; ICs0, 50% inhibitory concentration; DMSO, dimethylsulfoxide;BE benzo(a)pyrene
Abbreviations:
C o r r e s p o n d e n c e to: Nancy A. Lorr, Dept. Avian & Aquatic Animal Medicine, CornellUniversity,203 Rice Hall, Ithaca, NY 14853, USA
P450I are much higher in the supporting cells of immune organs than in the immune cells themselves.
Key words: P450I
Chicken
-
Immune tissue - Cytochrome
Introduction
I I
The sensitivity of an immune tissue to xenobiotic-induced damage may depend on the capacity of that immune tissue for metabolic activation of a particular toxin to forms which react with DNA and other macromolecules. The hepatic cytochrome P450 monooxygenase system oxidizes xenobiotics and endogenous substrates, transforming some molecules to reactive electrophilic intermediates. While some electrophilic intermediates are sufficiently stable to be transported from the liver to a target organ, or are carded from the liver in a transport form, the high reactiv-! ity and instability of some P450 metabolites may preclude translocation to the target organ. In the latter cases, target organ P450 may be responsible for toxicity. P450IA1 is induced by polycyclic aromatic hydrocarbons (e. g. 3-methylcholanthrene) and coplanar chlorinated hydrocarbons (e. g. 2,3,7,8-tetrachlorodibenzodioxin (TCDD)), and it also metabolizes most of these inducers. Basal and induced P450IA1, at low levels, has been detected in a variety of immune tissues including cultured human lymphocytes (Whitlock et al. 1972; Amsbaugh et al. 1986), macrophages from several species (Peters0n 1987), murine spleen (Blank et al. 1987; Kawabata and White, Jr. 1989) and rabbit bone marrow (Schnier et al. 1989). P450I-catalyzed activity was detected in the chicken thymus (Nikolaidis et al. 1988b; Misra et al. 1991) and bursa (Misra et al. 1991) after pretreatment with P4501 inducers. The P450I(s) form in chicken liver is similar to both P450IA1 and P450IA2 in mammals in its catalytic activity and inducibility (Hokama et al. 1988; Jacobs et a/.
561 Table 1. EROD and BZOD in microsomes from liver and lymphoid organs from control and induced 4 week old chickens. 1 EROD and BZOD is in prnol/mgmicrosomal protein/min. Values are means + standard deviations; the numbers in parentheses are the numbers of samples Organ2
Treatment
EROD
Liver
Corn Oil TCB Water PB
89.9 _+ 19.0 6363.3 • 1264.6 139.6 • 1.5 61.7 ___ 4.2
Bursa
Corn oil TCB
0.0 7.2 •
2.5
(5) (5)e
0.0 1.8 ...
Corn oil TCB
0.0 28.3 ___
9.5
(5) (5)b
0.0 4.6
(1) (1)
Corn oil TCB
0.0 1.3 ___
0.9
(4) (4)e
0.0 0.0
(1) (1)
Thymus PECs
BZOD (6) (12)a* (2) (2)
26.4 _ 6.2 1297.8 "+"254.8 33.7 _+ 0.3 20.5 + 0.3 0.6
(4) (3) (2) (2) (2) (2)
I Assaysrun with I p.M ethoxyresorufin and 5 I.tM benzyloxyphenoxazone 2 Microsomal preparations of liver, bursa, and thymus were made directlyfrom whole organ sonicated homogenates. Microsomal preparationsfrom PECs were made from washed and sonicated peritoneal exu-
date cells. All samples were stored frozen at -80 ~C * In comparing TCB-induced EROD of microsomes from different tissues, ERODs are statistically different (p
