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Immunocytotoxicity effects of polycyclic aromatic hydrocarbons on mouse lymphocytes
Toxicology, 31 (1984) 181-189 Elsevier ScientificPublishers Ireland Ltd.
IMMUNOCYTOTOXICITY E F F E C T S O F P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S ON MOUSE LYMPHOCYTES*
ARISTOWOJDANI,MEI-~DADATTARZADEH,GIRMAWOLDE.TSADIKand LAWRENCEJ. ALFREDt Departments of Pathology (L.J.A.) and Medicine (G. W-T.), Charles R. Drew Medical School and UCLA Cancer Center (L.J.A.) Los Angeles, CA (U.S.A.)
(Received September 7th, 1983) (Accepted February 2nd, 1984)
SUMMARY The in vivo effects of 3 polycyclic aromatic hydrocarbons (PAH): 3methylcholanthrene (IVICA), benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) on the ability of mouse lymphocytes to bind and kill target tumor cells in vitro were measured. C57 and C3H inbred mice were preiwmunized with P815 tumor cells and then treated with a single i.p. injection of corn oil alone or with varying doses of the above PAH compounds (0.5-50 mg/kg body wt). At different post-injection times, antigen sensitized splenic lymphocytes (SL) and peritoneal exudate lymphocytes (PEL) were measured for binding and killing rates, using a single cell assay. MCA doses of 5 and 50 mg/kg inhibited SL: target cell binding 29-42% and PEL: target cell binding 23-60%. BaP had a similar significant dose dependent suppression on SL and PEL binding. Target cell killing rates by SL and PEL from MCA and BaP treated C57 and C3H mice were consistently suppressed at significant levels, compared to oil injected controls (P < 0.05). On the other hand, binding and killing rates by SL and PEL from BeP treated mice showed an inconsistent and borderline significance at the above dose levels. When measured as a function of post-injection time of MCA, binding rates of SL from both mouse strains remained essentially unchanged after 10, 30 and 45 days. Target cell *This research was supported by grants from the EnvironmentalProtection Agency(R-807046), NIH/MBRS (RR-08140) and NIGMS/MARC(GM07971). tAddress all correspondence to: Lawrence Alfred, Ph.D., Charles R. Drew Medical School, 1621 East 120th Street Los Angeles, CA 90059, U.S.A. Abbreviations: BaP, benzo(a)pyrene; BeP, benzo(e)pyrene; CM, complete medium; CML, cellmediated lympholysis;IL-2, interleukin-2;LT, lymphotoxin;MCA,3-methylcholanthrene;PAH, polycyclic aromatic hydrocarbons; PEL, periteneal exudate lymphocytes; SL, splenic lymphocytes. 0300-483X/84/$03.00 © 1984 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
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killing by SL from C3H and C57 mice, however, was suppressed 55-65% after 10-30 days post-injection. At 45 days post-injection, the capacity of SL to kill target tumor cells was restored to 64-70% of control values. The results suggest that binding is an early event that depends on dose, whereas target cell killing is a function of dose and post-injection time.
Key words: Immunosuppression; P A H compounds; T-lymphocytes INTRODUCTION The mechanism of action of P A H compounds on the functions of cytotoxic T-lymphocytes have not been described. These substances have been shown to depress T-cell functions including blastogenesis [1], homograft reaction [2], graft vs. host reactivity [3], N K cell activity [4] and lymphotoxin (LT) production [5]. P A H compounds are ubiquitous environmental chemicals, are bioactivated via the mixed-function oxidases, and certain species are metabolized to toxic, mutagenic and carcinogenic metabolites [6-9]. A clear relationship has been established between P A H metabolism and tumor development in mice [10]. However, little attention has been given to early PAH-induced alterations in host effector cells which recognize new cell surface antigens and which are involved in the destruction of preneeplastic and tumor cells. Recent studies in this laboratory suggested that an MCA dose related suppression of blastogenesis and antigen specific cell-mediated lympholysis (CML) may be correlated with an increase in mouse splenic T-suppressor cells [1]. In the present study, we employed a single cell assay [11] to analyze early in vivo effects of MCA, B a P and BeP on the binding and lytic steps of CML. These P A H compounds were selected for this study because of their different carcinogenic activities. For example, MCA and B a P are potent carcinogens, while BeP is almost inactive [12]. Epoxides of BeP are weakly mutagenic in both bacterial and mammalian systems, compared to the high mutagenic activity of diolepoxides of B a P [13]. Therefore, it is of importance to relate immunocytotoxicity to carcinogenic potency of P A H compounds. MATERIALS AND METHODS
Mice Eight-week-old inbred strains of mice used in this investigation were C3H]fCUM, C57BL/6 CUM, purchased from the University of California at Berkeley. MCA, B a P and B e P were obtained from Sigma Chemical Company, St. Louis, MO. These mouse strains were used as a source of splenic effector cells because both" are P A H responsive with high tumorigenicity to Ba_P and MCA [10].
182
Target cells Tumor target cells were P815, syngeneic in DBA/2 (H-2d). This tumor was maintained in ascites form by serial passage via i.p. injection and passed on nylon wool columns before use as previously described [14].
Treatment of mice with PAIl compounds and preparation of immune lymphocytes Ten days prior to P A H treatment, mice were injected i.p. with 2 × 107 viable P815 cells for the production of immune lymphocytes. Mice were then singularly injected i.p. with varying doses of MCA, B a P or BeP in corn oil (0-50 mg/kg body wt). Twenty-four hours after t r e a t m e n t with P A H compounds, splenic lymphocytes (SL) and peritoneal exudate lymphocytes (PEL) from pooled spleens and peritoneal washings of 6 mice of each strain in each experiment were isolated as previously described [15]. P E L and SL were washed with H a n k s balanced salt solution (HBSS), resuspended at 15 × 10s cells/ml of a complete medium (CM) consisting of RPMI-1640 supplemented with 10% fetal calf serum, and were then placed onto a presoaked nylon wool column for I h at 37°C as described by Julius et al. [14]. Non-adherent mononuclear cells were eluted and the total number was determined with a Coulter Counter. The percentage of contaminating macrophages in nonadherent cell fractions was between 3% and 5% as shown using non-specific esterase staining [16]. In certain experiments, SL from MCA treated mice (50 mg/kg body wt) were isolated after 1, 10, 30 and 45 days. These SL were assayed for CML as described below. During treatment there was no mortality or weight loss in control or treated animals.
Single cell assay Conjugate formation and target cell killing was assayed as previously described [11]. Briefly, 3 x 108/mi of effector cells and 3 x 10S/ml of P815 were individually equilibrated in 30°C water bath. After equilibration, 100 p l of effector cells and 100 p l targets (E:T = 1:1) were mixed in glass test tubes, incubated at 30°C for. 5 rain and centrifuged for 5 rain at 200 rev./min. Fifty microliters of media was added to resuspend cell pellets and 100 #1 of molten agarose at 39°C was addecl. Agarose cell suspensions were plated onto agarose precoated slides. Slides were submerged in CM for 3-4 h at 37°C in a 5% CO2 incubator. Following incubation, cells were stained with 0.2% trypan blue for 5 rain, fixed in 0.15% formaldehyde and slides were scored for conjugate formation and target cell killing (trypan blue uptake). Conjugate and target cell lysis (killing) rates were calculated by the following formulae: % conjugates --
No. lymphocytes bound to targets × 100 total No. lymphocytes (free and bound)
% target killed = No. dead lymphocyte bound targets x 100 total No. lymphocyte bound targets (live and dead) 183
A s s a y s on e f f e c t o r - t a r g e t cell b i n d i n g a n d t a r g e t cell k i l l i n g were c a r r i e d o u t b l i n d l y b y t h e s a m e t e c h n i c i a n . T r i p l i c a t e slides of cells f r o m control a n d t r e a t e d mice w e r e ceded w i t h a n u m b e r i n g s y s t e m a n d r e a d w i t h i n 2 d a y s of p r e p a r a t i o n . E a c h e x p e r i m e n t w a s c o n d u c t e d such t h a t t h e c o n j u g a t e s identified w a s t h e fixed n u m b e r of 200, a n d t h e r e f o r e t h e n u m b e r of cells e x a m i n e d to d e t e r m i n e t h e b i n d i n g r a t e s v a r i e d f r o m 800 to 3400. T h r e e i n d e p e n d e n t d e t e r m i n a t i o n s w e r e m a d e a t each P A H dose level.
Statistical methods S t a t i s t i c a l significance of t h e p e r c e n t b i n d i n g a n d p e r c e n t killing, in t h e c o m p a r i s o n of t r e a t m e n t a n d control groups, were e s t a b l i s h e d t h r o u g h t h e use of t h e one-sided test for proportions. Since 3 d e t e r m i n a t i o n s of r a t e s w e r e m a d e a t e a c h P A H dose level, t h e m i n i m u m r a t e of t h e control g r o u p w a s c o m p a r e d to t h e m a x i m u m r a t e of t h e t r e a t m e n t group. T h i s is a cons e r v a t i v e a p p r o a c h t h a t lends s t r e n g t h to t h e s i g n i f i c a n t findings of t h e t r e a t m e n t a n d control c o m p a r i s o n s . Also, c o r r e l a t i o n a n a l y s i s of t h e b i n d i n g and killing rates were performed. RESULTS A single cell a s s a y w a s e m p l o y e d to m e a s u r e t h e effects of v a r y i n g doses of 3 P A H c o m p o u n d s on t h e b i n d i n g a n d k i l l i n g r a t e s of a n t i g e n specific C M L activity. B i n d i n g a n d k i l l i n g r a t e s b y SL a n d P E L isolated f r o m P A H t r e a t e d mice are s h o w n in T a b l e s I, I I a n d liT. E n t r i e s r e p r e s e n t t h e m e a n percen-
TABLE I EFFECTS OF MCA DOSE ON CONJUGATE FORMATION AND P815 TARGET CELL KILLING BY ANTIGEN SENSITIZED LYMPHOCYTES IN A SINGLE ASSAY~ Mouse strain C57
mg MCA/kg body wt
0
0.5 5.0 50.0 C3H
0 0.5 5.0 50.0
Splenic lymphocytes
Peritoneal exudate lymphocytes
% binding
% killing
% binding
% killing
12 ±
1.3 11 ± 1 8 ± 1.4" 7 ± 0.8*
42 ± 3.2 39 ± 5.4 25 ± 2.1" 15 ± 1.6"
18 ± 1.5 16 ± 1.7 14 ± 2* 11 ± 1.2"
48 ± 2.2 44 ± 2.8 21 ± 1.4" 13 -- 1.1"
14--- 1.7 12 ± 1.2 10 - 2.0* 9 ± 1.4"
44± 1.8 42 ± 3.7 26 ± 2.9* 18 ± 1.9"
20±3 23 ± 2 12 ± 1.6" 8 ± 0.9*
46± 1.7 39 ± 2.8 17 ± 1.2" 15 ± 1.6"
°Percent effector:target cell binding and target cell killing by MCA were measured in a single cell assay [11]. In each experiment, varying numbers of cells were examined until 200 conjugates were identified. The killing rates were based on this fixed number of conjugates. The table entries represent the mean percentages ± S.D. of 3 independent determinations. *Significant at the P = 0.05 level in treatment and centro~ comparison. 184
TABLE II EFFECT OF BaP ON CONJUGATE FORMATION AND TARGET CELL. KILLING BY ANTIGEN SENSITIZED LYMPHOCYTES~ Splenic lymphocytes
Peritoneal exudato lymphocytes
Mouse strain
m g BaP/kg body wt
% binding
% killing
% binding
% killing
C57
0 0.5 5 50
12-+1.3 10±1.7 7-+0.6* 6-+0.8*
5-+3.2 ~ -+4.6 21-+1.7" 13±1.1"
18±1.5 19±1.7 11-+1.6" 9±1.3"
~±2.2 46-+3.8 28±3.2* 16±L7"
C3H
0 0.5 5 50
14-+1.7 13±1.5 7-+0.8* 8-+1.2"
~-+1.8 46±1.6 19-+1.4" 15-+1.9"
20±3 16±2.1 9-+1.3" 6-+0.7*
46 -+1.7 41-+3.1 ~-+1.1" 21-+1.4"
aSee legend of Table I. TABLE HI EFFECT OF BeP ON CONJUGATE FORMATION AND TARGET CELL KILLING BY ANTIGEN SENSITIZED LYMPHOCYTES•
Splenic lymphocytes
Peritoneal exudato lymphocytos
Mouse strain
mg BeP/kg body wt
% binding
% killing
% binding
% killing
C57
0 0.5 5 50
12-+1.3 12-+1.1 11-+1.6 9+-1.2"
5-+3.2 ~±2.8 38-+2.3 ~ ±1.8
18-+1.5 17-+1.6 14±1.3" 15-+1.8"
46±2.2 46 -+3.8 ~ ±2.9t 39-+3.3t
C3H
0 0.5 5 50
14-+1.7 15 +-1.8 11 -+1.3" 12 +-2.1t
44-+1.8 42 -+2.7 3.9+ - 1.6 35 -+2.9*
20-+3 19-+ 1.7 17+- 2.2t 17 -+3.6t
46-+1.7 39 -+2.9 38-+ 3.4t 37 -+2.3*
"See legend of Table I. tBorderline significance at the P = 0.05 level (control vs. treatment).
t a g e s of 3 i n d e p e n d e n t d e t e r m i n a t i o n s a n d s t a n d a r d d e v i a t i o n s f o r measurements taken 24h after PAH treatment. Results summarized in T a b l e I s h o w t h a t a n M C A dose of 0.5 m g / k g b o d y w t h a d n o effect o n t h e b i n d i n g a n d l y t i c ( k i l l i n g ) s t e p s of C M L . M C A d o s e s of 5 a n d 50 m g / k g b o d y w t m g n i f i c a n t l y s u p p r e s s e d t h e b i n d i n g of S L ( 2 9 - 4 2 % ) a n d P E L ( 2 3 - 6 0 % ) . T a r g e t cell k i l l i n g b y S L w a s s u p p r e s s e d 4 0 - 6 5 % c o m p a r e d to a 5 7 - 7 3 % s u p p r e s s i o n of P E L . R e s u l t s s h o w n i n T a b l e I I d e m o n s t r a t e t h a t B a P h a d a v e r y s i m i l a r a n d c o n s i s t e n t d o s e - d e p e n d e n t s u p p r e s s i v e effect o n t h e a b i l i t y 185
of SL and PEL to bind and kill target tumor cells. In comparison, BeP showed little or no suppression on effector :target cell binding and of target cell killing a t doses of 0.5 mg/kg body wt. At a BeP dose of 0.5-50 mg/kg body wt, binding (15-25%) and target cell killing (15-20%) were suppressed in an inconsistent fashion, with frequent borderline significance (Table HI). MCA binding/killing correlations were determined for all dose levels in both SL and PEL. C57 treated mice showed correlations of 0.85 and 0.86 for SL and PEL, while binding/killing correlations for C3H treated mice were 0.84 and 0.89 for SL and PEL. These values for 24-h pest-injection time indicate a strong association of an inhibition in conjugate formation and suppression of target cell killing. The effects of MCA on effector:target cell binding and target cell lysis were measured as a function of time after injection. Results shown in Table IV illustrate t h a t 10, 30 and 45 days post-injection with 50 mg/kg body wt, binding was inhibited 28%, 42% and 10%, respectively, by splenic lymphocytes from C57 mice. Binding by C3H mouse lymphocytes was inhibited 24%, 34% and 10% during these periods. The ability of C57 splenic lymphocytes to kill target cells at 10, 30 and 45 days post-treatment was inhibited 70%, 50% and 23%, compared to an inhibition of 63%, 55% and 36% killing by C3H splenic lymphocytes. The capacity of SL to bind and kill target cells increased from 8% to 10% at 10 days post MCA t r e a t m e n t to 27-30% after 45 days. DISCUSSION
Immunosuppression induced in experimental animals by chemical carcinogens is thought to play a major role in malignant t u m o r development in
TABLE IV EFFECTS OF TIME ON PERCENT CONJUGATE FORMATION AND KILLING BY SPLENIC LYMPHOCYTESFROMCONTROLAND MCA TREATED MICE Days post-injection I0 Mouse strain
mg MCA/Kg body wt
30
45
% binding % killing % binding % killing % binding % killing
C57
0 50
11-+0.9 8_+1.1a
43_+3.6 12+1.6 13_+1.7* 7-+0.4*
C3H
0 50
13_+1.3 41_+1.9 12-+1.1 40-+1.9 11-+1.7 42-+2 10_+0.8" 15_+1.4a 8+_0.9~ 18_+1.3" 10_+0.5 2 7 + 3 . 3
"Significantat 0.05 level (dose comparison at fixed time). bSignificantat 0.05 level (time comparisonat fixed dose). 186
41_+2.3 10_+1.2 39_+3.1 16_+1.8" 9_+0.6 3 0 - + 2 . 4 ~'b *'b
these hosts [17,18]. In the current study we employed a single cell assay to measure direct in vivo cytotoxic action of PAH compounds on the binding and killing steps of CML in vitro. In mice injected with MCA or BaP at doses equal to or greater than 5 mg/kg body wt, splenic and peritoneal exudate effector lymphocytes show a statistically significant decrease in binding and killing of target cells. This consistent and significant decrease in effector :target cell conjugate formation is associated with decreases in target cell killing. These observations further confirm recent reports by Bonavida et al. [19] on the relationship between binding and lysis in cell-mediated cytotoxicity. There is a high correlation between binding and killing rates of SL and PEL isolated from mice treated with MCA for 24 h. On the other hand, the effect of BeP on binding and killing of target cells shows less consistency and also suggests that the dose (50 mg/kg body wt) causing a significant decrease in CML is higher than those for MCA or BaP. This observation on the different levels of cytotoxicity of MCA, BaP and BeP is consistent with existing knowledge of the relative carcinogenicity of these compounds [12,20]. That is, BeP is only weakly activated to dihydrodiols and diolepoxides [21], and low concentrations of these metabolites may be correlated with low levels of toxicity of this PAH compound. The formation of conjugates as a function of post-injection time for control and MCA treated (50 mg/kg body wt) mice indicates that binding rates do not significantly change with time. Also, the killing rates for effector cells from control mice do not show any appreciable change with time. However, the killing rates of lymphocytes from MCA treated animals increase with postinjection time, reaching statistically significant levels after 30 days. Hence, high binding and killing correlations noted above in M C A treated mice is an early phenomenon. These findings suggest that the in vivo M C A induced inhibition of effector cell binding to target cells is dose-dependent but is independent of time. Target cellkilling,however, appears to be both dose and time dependent. The M C A induced suppression of the ability of splenic effector lymphocytes to kill target tumor cells,as measured in an antigen specific single cell assay, is restored with time (Table IV). This is not the case with P A H induced suppression of N K cell activity which can be transiently restored by biological response modifiers [18]. Recent findings in this laboratory suggest a relationship between an augmentation of splenic N K cell activity by C. p a r v u m injection and a delay in the arise of palable M C A induced tumors (Alfred et al., unpublished). M a n y investigators agree that the first line of natural i m m u n e defense is believed to be N K cell activity [21,22], and this process appears to be irreversibly inhibited by P A H carcinogens.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr. Benjamin Bonavida, Immunology and Microbiology Department, UCLA School of Medicine, for providing us 187
w i t h t h e P 8 1 5 t a r g e t cells. W e a r e i n d e b t e d to Ms. P a t r i c e H a m i l t o n a s s i s t a n c e i n t h e p r e p a r a t i o n of t h i s m a n u s c r i p t .
for
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IMMUNOCYTOTOXICITY E F F E C T S O F P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S ON MOUSE LYMPHOCYTES*
ARISTOWOJDANI,MEI-~DADATTARZADEH,GIRMAWOLDE.TSADIKand LAWRENCEJ. ALFREDt Departments of Pathology (L.J.A.) and Medicine (G. W-T.), Charles R. Drew Medical School and UCLA Cancer Center (L.J.A.) Los Angeles, CA (U.S.A.)
(Received September 7th, 1983) (Accepted February 2nd, 1984)
SUMMARY The in vivo effects of 3 polycyclic aromatic hydrocarbons (PAH): 3methylcholanthrene (IVICA), benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) on the ability of mouse lymphocytes to bind and kill target tumor cells in vitro were measured. C57 and C3H inbred mice were preiwmunized with P815 tumor cells and then treated with a single i.p. injection of corn oil alone or with varying doses of the above PAH compounds (0.5-50 mg/kg body wt). At different post-injection times, antigen sensitized splenic lymphocytes (SL) and peritoneal exudate lymphocytes (PEL) were measured for binding and killing rates, using a single cell assay. MCA doses of 5 and 50 mg/kg inhibited SL: target cell binding 29-42% and PEL: target cell binding 23-60%. BaP had a similar significant dose dependent suppression on SL and PEL binding. Target cell killing rates by SL and PEL from MCA and BaP treated C57 and C3H mice were consistently suppressed at significant levels, compared to oil injected controls (P < 0.05). On the other hand, binding and killing rates by SL and PEL from BeP treated mice showed an inconsistent and borderline significance at the above dose levels. When measured as a function of post-injection time of MCA, binding rates of SL from both mouse strains remained essentially unchanged after 10, 30 and 45 days. Target cell *This research was supported by grants from the EnvironmentalProtection Agency(R-807046), NIH/MBRS (RR-08140) and NIGMS/MARC(GM07971). tAddress all correspondence to: Lawrence Alfred, Ph.D., Charles R. Drew Medical School, 1621 East 120th Street Los Angeles, CA 90059, U.S.A. Abbreviations: BaP, benzo(a)pyrene; BeP, benzo(e)pyrene; CM, complete medium; CML, cellmediated lympholysis;IL-2, interleukin-2;LT, lymphotoxin;MCA,3-methylcholanthrene;PAH, polycyclic aromatic hydrocarbons; PEL, periteneal exudate lymphocytes; SL, splenic lymphocytes. 0300-483X/84/$03.00 © 1984 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
181
killing by SL from C3H and C57 mice, however, was suppressed 55-65% after 10-30 days post-injection. At 45 days post-injection, the capacity of SL to kill target tumor cells was restored to 64-70% of control values. The results suggest that binding is an early event that depends on dose, whereas target cell killing is a function of dose and post-injection time.
Key words: Immunosuppression; P A H compounds; T-lymphocytes INTRODUCTION The mechanism of action of P A H compounds on the functions of cytotoxic T-lymphocytes have not been described. These substances have been shown to depress T-cell functions including blastogenesis [1], homograft reaction [2], graft vs. host reactivity [3], N K cell activity [4] and lymphotoxin (LT) production [5]. P A H compounds are ubiquitous environmental chemicals, are bioactivated via the mixed-function oxidases, and certain species are metabolized to toxic, mutagenic and carcinogenic metabolites [6-9]. A clear relationship has been established between P A H metabolism and tumor development in mice [10]. However, little attention has been given to early PAH-induced alterations in host effector cells which recognize new cell surface antigens and which are involved in the destruction of preneeplastic and tumor cells. Recent studies in this laboratory suggested that an MCA dose related suppression of blastogenesis and antigen specific cell-mediated lympholysis (CML) may be correlated with an increase in mouse splenic T-suppressor cells [1]. In the present study, we employed a single cell assay [11] to analyze early in vivo effects of MCA, B a P and BeP on the binding and lytic steps of CML. These P A H compounds were selected for this study because of their different carcinogenic activities. For example, MCA and B a P are potent carcinogens, while BeP is almost inactive [12]. Epoxides of BeP are weakly mutagenic in both bacterial and mammalian systems, compared to the high mutagenic activity of diolepoxides of B a P [13]. Therefore, it is of importance to relate immunocytotoxicity to carcinogenic potency of P A H compounds. MATERIALS AND METHODS
Mice Eight-week-old inbred strains of mice used in this investigation were C3H]fCUM, C57BL/6 CUM, purchased from the University of California at Berkeley. MCA, B a P and B e P were obtained from Sigma Chemical Company, St. Louis, MO. These mouse strains were used as a source of splenic effector cells because both" are P A H responsive with high tumorigenicity to Ba_P and MCA [10].
182
Target cells Tumor target cells were P815, syngeneic in DBA/2 (H-2d). This tumor was maintained in ascites form by serial passage via i.p. injection and passed on nylon wool columns before use as previously described [14].
Treatment of mice with PAIl compounds and preparation of immune lymphocytes Ten days prior to P A H treatment, mice were injected i.p. with 2 × 107 viable P815 cells for the production of immune lymphocytes. Mice were then singularly injected i.p. with varying doses of MCA, B a P or BeP in corn oil (0-50 mg/kg body wt). Twenty-four hours after t r e a t m e n t with P A H compounds, splenic lymphocytes (SL) and peritoneal exudate lymphocytes (PEL) from pooled spleens and peritoneal washings of 6 mice of each strain in each experiment were isolated as previously described [15]. P E L and SL were washed with H a n k s balanced salt solution (HBSS), resuspended at 15 × 10s cells/ml of a complete medium (CM) consisting of RPMI-1640 supplemented with 10% fetal calf serum, and were then placed onto a presoaked nylon wool column for I h at 37°C as described by Julius et al. [14]. Non-adherent mononuclear cells were eluted and the total number was determined with a Coulter Counter. The percentage of contaminating macrophages in nonadherent cell fractions was between 3% and 5% as shown using non-specific esterase staining [16]. In certain experiments, SL from MCA treated mice (50 mg/kg body wt) were isolated after 1, 10, 30 and 45 days. These SL were assayed for CML as described below. During treatment there was no mortality or weight loss in control or treated animals.
Single cell assay Conjugate formation and target cell killing was assayed as previously described [11]. Briefly, 3 x 108/mi of effector cells and 3 x 10S/ml of P815 were individually equilibrated in 30°C water bath. After equilibration, 100 p l of effector cells and 100 p l targets (E:T = 1:1) were mixed in glass test tubes, incubated at 30°C for. 5 rain and centrifuged for 5 rain at 200 rev./min. Fifty microliters of media was added to resuspend cell pellets and 100 #1 of molten agarose at 39°C was addecl. Agarose cell suspensions were plated onto agarose precoated slides. Slides were submerged in CM for 3-4 h at 37°C in a 5% CO2 incubator. Following incubation, cells were stained with 0.2% trypan blue for 5 rain, fixed in 0.15% formaldehyde and slides were scored for conjugate formation and target cell killing (trypan blue uptake). Conjugate and target cell lysis (killing) rates were calculated by the following formulae: % conjugates --
No. lymphocytes bound to targets × 100 total No. lymphocytes (free and bound)
% target killed = No. dead lymphocyte bound targets x 100 total No. lymphocyte bound targets (live and dead) 183
A s s a y s on e f f e c t o r - t a r g e t cell b i n d i n g a n d t a r g e t cell k i l l i n g were c a r r i e d o u t b l i n d l y b y t h e s a m e t e c h n i c i a n . T r i p l i c a t e slides of cells f r o m control a n d t r e a t e d mice w e r e ceded w i t h a n u m b e r i n g s y s t e m a n d r e a d w i t h i n 2 d a y s of p r e p a r a t i o n . E a c h e x p e r i m e n t w a s c o n d u c t e d such t h a t t h e c o n j u g a t e s identified w a s t h e fixed n u m b e r of 200, a n d t h e r e f o r e t h e n u m b e r of cells e x a m i n e d to d e t e r m i n e t h e b i n d i n g r a t e s v a r i e d f r o m 800 to 3400. T h r e e i n d e p e n d e n t d e t e r m i n a t i o n s w e r e m a d e a t each P A H dose level.
Statistical methods S t a t i s t i c a l significance of t h e p e r c e n t b i n d i n g a n d p e r c e n t killing, in t h e c o m p a r i s o n of t r e a t m e n t a n d control groups, were e s t a b l i s h e d t h r o u g h t h e use of t h e one-sided test for proportions. Since 3 d e t e r m i n a t i o n s of r a t e s w e r e m a d e a t e a c h P A H dose level, t h e m i n i m u m r a t e of t h e control g r o u p w a s c o m p a r e d to t h e m a x i m u m r a t e of t h e t r e a t m e n t group. T h i s is a cons e r v a t i v e a p p r o a c h t h a t lends s t r e n g t h to t h e s i g n i f i c a n t findings of t h e t r e a t m e n t a n d control c o m p a r i s o n s . Also, c o r r e l a t i o n a n a l y s i s of t h e b i n d i n g and killing rates were performed. RESULTS A single cell a s s a y w a s e m p l o y e d to m e a s u r e t h e effects of v a r y i n g doses of 3 P A H c o m p o u n d s on t h e b i n d i n g a n d k i l l i n g r a t e s of a n t i g e n specific C M L activity. B i n d i n g a n d k i l l i n g r a t e s b y SL a n d P E L isolated f r o m P A H t r e a t e d mice are s h o w n in T a b l e s I, I I a n d liT. E n t r i e s r e p r e s e n t t h e m e a n percen-
TABLE I EFFECTS OF MCA DOSE ON CONJUGATE FORMATION AND P815 TARGET CELL KILLING BY ANTIGEN SENSITIZED LYMPHOCYTES IN A SINGLE ASSAY~ Mouse strain C57
mg MCA/kg body wt
0
0.5 5.0 50.0 C3H
0 0.5 5.0 50.0
Splenic lymphocytes
Peritoneal exudate lymphocytes
% binding
% killing
% binding
% killing
12 ±
1.3 11 ± 1 8 ± 1.4" 7 ± 0.8*
42 ± 3.2 39 ± 5.4 25 ± 2.1" 15 ± 1.6"
18 ± 1.5 16 ± 1.7 14 ± 2* 11 ± 1.2"
48 ± 2.2 44 ± 2.8 21 ± 1.4" 13 -- 1.1"
14--- 1.7 12 ± 1.2 10 - 2.0* 9 ± 1.4"
44± 1.8 42 ± 3.7 26 ± 2.9* 18 ± 1.9"
20±3 23 ± 2 12 ± 1.6" 8 ± 0.9*
46± 1.7 39 ± 2.8 17 ± 1.2" 15 ± 1.6"
°Percent effector:target cell binding and target cell killing by MCA were measured in a single cell assay [11]. In each experiment, varying numbers of cells were examined until 200 conjugates were identified. The killing rates were based on this fixed number of conjugates. The table entries represent the mean percentages ± S.D. of 3 independent determinations. *Significant at the P = 0.05 level in treatment and centro~ comparison. 184
TABLE II EFFECT OF BaP ON CONJUGATE FORMATION AND TARGET CELL. KILLING BY ANTIGEN SENSITIZED LYMPHOCYTES~ Splenic lymphocytes
Peritoneal exudato lymphocytes
Mouse strain
m g BaP/kg body wt
% binding
% killing
% binding
% killing
C57
0 0.5 5 50
12-+1.3 10±1.7 7-+0.6* 6-+0.8*
5-+3.2 ~ -+4.6 21-+1.7" 13±1.1"
18±1.5 19±1.7 11-+1.6" 9±1.3"
~±2.2 46-+3.8 28±3.2* 16±L7"
C3H
0 0.5 5 50
14-+1.7 13±1.5 7-+0.8* 8-+1.2"
~-+1.8 46±1.6 19-+1.4" 15-+1.9"
20±3 16±2.1 9-+1.3" 6-+0.7*
46 -+1.7 41-+3.1 ~-+1.1" 21-+1.4"
aSee legend of Table I. TABLE HI EFFECT OF BeP ON CONJUGATE FORMATION AND TARGET CELL KILLING BY ANTIGEN SENSITIZED LYMPHOCYTES•
Splenic lymphocytes
Peritoneal exudato lymphocytos
Mouse strain
mg BeP/kg body wt
% binding
% killing
% binding
% killing
C57
0 0.5 5 50
12-+1.3 12-+1.1 11-+1.6 9+-1.2"
5-+3.2 ~±2.8 38-+2.3 ~ ±1.8
18-+1.5 17-+1.6 14±1.3" 15-+1.8"
46±2.2 46 -+3.8 ~ ±2.9t 39-+3.3t
C3H
0 0.5 5 50
14-+1.7 15 +-1.8 11 -+1.3" 12 +-2.1t
44-+1.8 42 -+2.7 3.9+ - 1.6 35 -+2.9*
20-+3 19-+ 1.7 17+- 2.2t 17 -+3.6t
46-+1.7 39 -+2.9 38-+ 3.4t 37 -+2.3*
"See legend of Table I. tBorderline significance at the P = 0.05 level (control vs. treatment).
t a g e s of 3 i n d e p e n d e n t d e t e r m i n a t i o n s a n d s t a n d a r d d e v i a t i o n s f o r measurements taken 24h after PAH treatment. Results summarized in T a b l e I s h o w t h a t a n M C A dose of 0.5 m g / k g b o d y w t h a d n o effect o n t h e b i n d i n g a n d l y t i c ( k i l l i n g ) s t e p s of C M L . M C A d o s e s of 5 a n d 50 m g / k g b o d y w t m g n i f i c a n t l y s u p p r e s s e d t h e b i n d i n g of S L ( 2 9 - 4 2 % ) a n d P E L ( 2 3 - 6 0 % ) . T a r g e t cell k i l l i n g b y S L w a s s u p p r e s s e d 4 0 - 6 5 % c o m p a r e d to a 5 7 - 7 3 % s u p p r e s s i o n of P E L . R e s u l t s s h o w n i n T a b l e I I d e m o n s t r a t e t h a t B a P h a d a v e r y s i m i l a r a n d c o n s i s t e n t d o s e - d e p e n d e n t s u p p r e s s i v e effect o n t h e a b i l i t y 185
of SL and PEL to bind and kill target tumor cells. In comparison, BeP showed little or no suppression on effector :target cell binding and of target cell killing a t doses of 0.5 mg/kg body wt. At a BeP dose of 0.5-50 mg/kg body wt, binding (15-25%) and target cell killing (15-20%) were suppressed in an inconsistent fashion, with frequent borderline significance (Table HI). MCA binding/killing correlations were determined for all dose levels in both SL and PEL. C57 treated mice showed correlations of 0.85 and 0.86 for SL and PEL, while binding/killing correlations for C3H treated mice were 0.84 and 0.89 for SL and PEL. These values for 24-h pest-injection time indicate a strong association of an inhibition in conjugate formation and suppression of target cell killing. The effects of MCA on effector:target cell binding and target cell lysis were measured as a function of time after injection. Results shown in Table IV illustrate t h a t 10, 30 and 45 days post-injection with 50 mg/kg body wt, binding was inhibited 28%, 42% and 10%, respectively, by splenic lymphocytes from C57 mice. Binding by C3H mouse lymphocytes was inhibited 24%, 34% and 10% during these periods. The ability of C57 splenic lymphocytes to kill target cells at 10, 30 and 45 days post-treatment was inhibited 70%, 50% and 23%, compared to an inhibition of 63%, 55% and 36% killing by C3H splenic lymphocytes. The capacity of SL to bind and kill target cells increased from 8% to 10% at 10 days post MCA t r e a t m e n t to 27-30% after 45 days. DISCUSSION
Immunosuppression induced in experimental animals by chemical carcinogens is thought to play a major role in malignant t u m o r development in
TABLE IV EFFECTS OF TIME ON PERCENT CONJUGATE FORMATION AND KILLING BY SPLENIC LYMPHOCYTESFROMCONTROLAND MCA TREATED MICE Days post-injection I0 Mouse strain
mg MCA/Kg body wt
30
45
% binding % killing % binding % killing % binding % killing
C57
0 50
11-+0.9 8_+1.1a
43_+3.6 12+1.6 13_+1.7* 7-+0.4*
C3H
0 50
13_+1.3 41_+1.9 12-+1.1 40-+1.9 11-+1.7 42-+2 10_+0.8" 15_+1.4a 8+_0.9~ 18_+1.3" 10_+0.5 2 7 + 3 . 3
"Significantat 0.05 level (dose comparison at fixed time). bSignificantat 0.05 level (time comparisonat fixed dose). 186
41_+2.3 10_+1.2 39_+3.1 16_+1.8" 9_+0.6 3 0 - + 2 . 4 ~'b *'b
these hosts [17,18]. In the current study we employed a single cell assay to measure direct in vivo cytotoxic action of PAH compounds on the binding and killing steps of CML in vitro. In mice injected with MCA or BaP at doses equal to or greater than 5 mg/kg body wt, splenic and peritoneal exudate effector lymphocytes show a statistically significant decrease in binding and killing of target cells. This consistent and significant decrease in effector :target cell conjugate formation is associated with decreases in target cell killing. These observations further confirm recent reports by Bonavida et al. [19] on the relationship between binding and lysis in cell-mediated cytotoxicity. There is a high correlation between binding and killing rates of SL and PEL isolated from mice treated with MCA for 24 h. On the other hand, the effect of BeP on binding and killing of target cells shows less consistency and also suggests that the dose (50 mg/kg body wt) causing a significant decrease in CML is higher than those for MCA or BaP. This observation on the different levels of cytotoxicity of MCA, BaP and BeP is consistent with existing knowledge of the relative carcinogenicity of these compounds [12,20]. That is, BeP is only weakly activated to dihydrodiols and diolepoxides [21], and low concentrations of these metabolites may be correlated with low levels of toxicity of this PAH compound. The formation of conjugates as a function of post-injection time for control and MCA treated (50 mg/kg body wt) mice indicates that binding rates do not significantly change with time. Also, the killing rates for effector cells from control mice do not show any appreciable change with time. However, the killing rates of lymphocytes from MCA treated animals increase with postinjection time, reaching statistically significant levels after 30 days. Hence, high binding and killing correlations noted above in M C A treated mice is an early phenomenon. These findings suggest that the in vivo M C A induced inhibition of effector cell binding to target cells is dose-dependent but is independent of time. Target cellkilling,however, appears to be both dose and time dependent. The M C A induced suppression of the ability of splenic effector lymphocytes to kill target tumor cells,as measured in an antigen specific single cell assay, is restored with time (Table IV). This is not the case with P A H induced suppression of N K cell activity which can be transiently restored by biological response modifiers [18]. Recent findings in this laboratory suggest a relationship between an augmentation of splenic N K cell activity by C. p a r v u m injection and a delay in the arise of palable M C A induced tumors (Alfred et al., unpublished). M a n y investigators agree that the first line of natural i m m u n e defense is believed to be N K cell activity [21,22], and this process appears to be irreversibly inhibited by P A H carcinogens.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr. Benjamin Bonavida, Immunology and Microbiology Department, UCLA School of Medicine, for providing us 187
w i t h t h e P 8 1 5 t a r g e t cells. W e a r e i n d e b t e d to Ms. P a t r i c e H a m i l t o n a s s i s t a n c e i n t h e p r e p a r a t i o n of t h i s m a n u s c r i p t .
for
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