Differential immunotoxic effects of the environmental chemical benzo[a]pyrene in young and aged mice

Mechanisms of Ageing and Development, 30 (1985) 333-341

333

Elsevier ScientificPublishersIreland Ltd.

Brief Note

DIFFERENTIAL IMMUNOTOXIC EFFECTS OF THE ENVIRONMENTAL CHEMICAL BENZO[a]PYRENE IN YOUNG AND AGED MICE MARK LYTE* and PETER H. BICK Department of Microbiology and Immunology, Medical College of Virginia. Box 6 78, MCV Station, Richmond, VA 23298-0001 (U.S.A.}

(Received October 15th, 1984) (Revisionreceived February 12th, 1985) SUMMARY Young (3-6 months), middle-aged (16-18 months) and aged (23-26 months) mice were exposed in vitro and in vivo to the immunotoxic environmental chemical benzo [a] pyrene. The generation of antibody producing cells to the T-dependent antigens of sheep erythrocytes was observed to be suppressed in all age groups. Significantly, aged mice were shown to exhibit a greater percent suppression of antibody responses than young or middle-aged mice both in vitro and in vivo. The results presented provide the first evidence that the degree of immunological toxicity of environmental chemicals may be partially dependent upon the chronological and immunological age of the animal.

K e y words: Aging; Benzo [a] pyrene; Immunotoxicity

INTRODUCTION Polycyclic aromatic daydrocarbons (PAHs), of which benzo[a] pyrene (BaP) is representative, constitute a family of chemicals which are highly prevalent in the environment and have been demonstrated to possess significant immunotoxic and carcinogenic properties [1-3]. The immunotoxic effects of PAl-ls were observed as early as 1952 when Malmgren et al. demonstrated that exposure of mice to 1,2,5,6-dibenzanthracene, 1,2benzanthracene and 3-methylcholanthrene resulted in a decrease in hemolytic antibody titers against sheep erythrocytes [2]. Treatment of mice with non-carcinogenic analogs did not, however, result in a reduction of hemolytic antibody titers. More recently, Dean et al. have reported suppression of IgM and IgG plaque forming responses to both Tdependent and T-independent antigens in mice exposed to BaP [3]. *To whom correspondenceshould be addressed. 004%6374/85/$03.30

Printed and Publishedin Ireland

© 1985 ElsevierScientificPublishersIreland Ltd.

334 The immunosuppressive effects of some environmental chemicals, including the polyaromatic hydrocarbons, are most pronounced in the developing immune system of the neonate [ 4 - 6 ] . Young adult mice ( 3 - 6 months) which possess a fully developed and functional immune system have been shown to be more refractory to PAH induced immunotoxicity than neonates. However, the imnrunotoxic effects of PAHs. or any environmental chemical, in aged mice have not yet been investigated. Since it has been well documented that a number of immune responses such as T cell proliferative responses. cell-mediated lymphocytotoxicity and generation of antibody producing cells decline with advancing age, it is important to examine the effects of BaP exposure of young and aged mice on immune competency [7,8]. In the present study we examine the generation of antibody producing cells to a Tdependent antigen in young ( 3 - 6 nronths), middle-aged (13 16 months)and aged (23 26 months) mice exposed in vitro and in vivo to BaP. The results presented provide the first evidence that an aged population may be at greater risk than young adults to the immunotoxicological effects of environmental chemicals such as BaP. MATERIALS AND METHODS

Mice (C57BL/6 X C3H/HeN)F1 (B6C3F1) male mice of different ages were obtained from the National Institutes of Aging colonies maintained by Charles River Breeding Laboratories, Kingston, NY. The mean life-span for this strain is approximately 30 months [9,10]. Mice were individually housed and fed chow and water ad libitum. A 12-hour light/dark cycle was maintained. Necropsy was performed on all mice used in the experimental systems described below. Individuals found to exhibit gross abnormalities were excluded from the study. Media and chemicals Rosewell Park Memorial Institute 1640 medium (RPMI) was purchased from Flow Laboratories, McClean, VA. Fetal bovine serum (FCS) was obtained from HyClone-Sterile Systems, Inc., Logan, UT. Sheep red blood cells (SRBC) were obtained from Colorado Serum Company, Denver, CO. Phosphate buffered saline (PBS)and NaC1 (0.85% NaC1 in distilled water) were prepared in the laboratory. Polyvinylpyrrolidone (PVP) of 10 000 molecular weight and benzo[a]pyrene (BaP) of approximately 98% purity were purchased from Sigma, St. Louis, MO. Gentamicin was purchased from Schering Pharmaceutical Corporation, Kenilworth, NJ. Pronase was obtained from Calbiochem-Behring Corp.,

La JoUa, CA. Preparation o f BaP solutions For & vitro studies BaP was suspended at desired concentrations in PVP-PBS medium (3.5 g PVP/100 ml PBS, pH 7.1) by vigorous stirring overnight at room temperature. For in vivo, studies BaP was suspended at 4 mg/ml of PVP/NaC1 medium (3.5 g PVP/100 ml

335 NaC1, pH 7.1) by vigorous stirring overnight at room temperature. All BaP preparations were used within 8 days and were kept at 4°C in the dark.

In vitro generation of anti-SRBC antibody forming cells Splenocytes were isolated from mice of varying ages on an individual basis by gently pressing the excised spleen between the frosted ends of two microscope slides. Splenocytes were adjusted to 5 × 106 spleen cells/ml of RPMI + 10% FCS + 50/ag gentamicin/ml. SRBC were added to yield a final ratio of 1 : 2 splenocytes: SRBC. RPMI alone (naive) or PVP-PBS (vehicle) or desired concentrations of BaP in PVP-PBS (test) were added in 0.1 ml doses/ml of splenocyte-SRBC suspension. Triplicate 1-ml cultures were plated in 24-well plates (Falcon No. 3072) and incubated for 4 - 5 days at 37°C in an atmosphere of 10% CO2, 7% 02 and 83% N2. Prior to enumeration of plaque forming cells the cultures were spun down in the 24-well plate at 100 g and washed once with RPMI alone. The viability of cultured cells was judged by phase contrast microscopy after treatment with pronase. Briefly, 0.1 ml of a 3.25 mg pronase/ml of saline solution was mixed with 0.1 ml cells and incubated for 5 min at 37°C. Ten milliliters of counting fluid was then added and cell counts performed on a Coulter Counter model ZBI following the method of Stewart [ 11 ]. Each culture was individually analysed for anti-SRBC producing cells.

In vivo generation of anti-SRBC antibody forming cells Mice of different ages were intraperitoneally (i.p.) dosed with 40 mg BaP in PVP/NaC1 per kg of body weight 4 days prior to and 4 days following i.p. immunization with 1 × lO s SRBC. No significant differences in body weights due to the dosing regimen were noted. Prior to enumeration of anti-SRBC antibody forming cells, the spleens were excised and splenocytes obtained by gently pressing the spleens between the frosted ends of two microscope slides. Each individual animal was assessed for anti-SRBC antibody forming spleen cells.

Enumeration of anti-SRBC antibody forming cells Direct lgM plaques against SRBC were enumerated by a modification of the Jerne technique [12]. One-tenth milliliter aliquots from each cell sample were added to tubes containing agar and sheep red blood cells maintained at 47°C. Complement (Sigma; diluted 1:4 in balanced salt solution) was then added, the tubes were thoroughly mixed and poured into petri dishes. Coverslips were gently placed on top of the mixtures to achieve a monolayer. Plates were incubated for 3 - 4 h at 37°C and plaques were counted using a plaque counter (Bellco).

Statistics All data were analyzed using the Bartlett's test for homogeneity and a one-way analysis of variance (ANOVA). If analysis of the data using ANOVA detected significant effects as a result of treatment, a Dunnett's t-test was performed if the data were found to be homogenous by the Bartlett's test. The Wilcoxon Rank test was used for non-homogenous data. All data are reported as the mean - standard error of the mean.

336 RESULTS In vitro BaP exposure

Splenocytes from individual naive young ( 3 - 6 months) and aged ( 2 3 - 2 6 months) mice were exposed in vitro to increasing concentrations of BaP. Pooling of spleen cells was avoided due to the potential presence of low and high responders in any one age group. This has been demonstrated to be of particular importance in the evaluation of of immune responsiveness in aged mice where the heterogeneity of immune responses between individual animals is much greater than observed in the young [14]. Figures 1 and 2 show the anti-SRBC plaque forming responses of splenocytes from 3- to 6-month (Fig. 1) and 23- to 26-month-old mice (Fig. 2). Naive and/or vehicle exposed splenocytes from aged mice evidenced a marked decrease in anti-SRBC producing capacity when compared to similiarly treated splenocytes from the young. The degree of reduction in plaque responses is in good agreement with previous reports [8,13-14]. It should be noted in Figs. 1 and 2 that the responses of vehicle treated splenocytes did not significantly differ from naive. Previous reports have demonstrated that PVP is a T-independent antigen and is capable of inducing non-specific polyclonal activation of B cells [15,16]. However, these reports utilized PVP preparations of greater than 350 000 molecular weight and in much higher concentrations than utilized in this study. Exposure of splenocytes from

5000 c/) -J -J

uJ o iii ,,_i >

4000

3000

c0 o

2000

o u_ a.

oooI NAIVE

VEHICLE

1

10

5'0

BaP (laglml)

Fig. 1. In vitro effect of BaP on anti-SRBC plaque formingresponses of splenocytesfrom 3- to 6-monthold mice. Splenocytes from individual mice were cultured with SRBC in the presence of media (naive), PVP (vehicle) or increasing concentrations of BaP (test) for 4-5 days and assayed for antibody formhag cells. Each line represents the plaque forming response of splenocytes from an individual mouse. Data shown is representative of 14 mice tested.

337 300

09

250

..J ..,.I

uJ 0 200. LU

m 150" o 100o I.L Q. 50

*---------,.

NAIVE

~=

VEHICLE

1

\

10

50

BaP (p.g/ml) Fig. 2. In vitro effect of BaP on anti-SRBC plaque forming responses of splenocytes from 23- to 26month-old mice. Splenocytes from individual mice were cultured with SRBC in the presence of media (naive), PVP (vehicle) or increasing concentrations of BaP (test) for 4 - 5 days and assayed ibr antibody producing cells. Each line represents the plaque forming response of splenoeytes from an individual mouse. Data shown is representative of 17 mice tested.

young mice to increasing concentrations of BaP resulted in a gradual suppression of antiSRBC activity (Fig. 1). Significantly, the dose response curves for similarly exposed splenocytes from the aged mice evidenced a greater degree of suppression that was induced at lower BaP concentrations (Figs. 2 and 3). The Percent suppression induced by increasing concentrations of BaP in 3 - 6 - and 23-26-month-old mice are shown in Fig. 3. No significant differences in cell numbers and viability were noted among the groups tested. In vivo BaP exposure

Three age groups of mice, 3 - 6 months, 16-18 months and 2 3 - 2 6 months, were exposed i.p. to 40 mg/kg BaP for 4 days prior to and for 4 days following immunization with 1 × 108 SRBC. This dosing regimen was chosen since it enabled the evaluation of the immunotoxic effects of BaP without involvement of the carcinogenic effects which result from chronic exposure. As shown in Table I an age related decline in IgM plaque forming responses from 3 - 6 months to 2 3 - 2 6 months is evident. No significant differences in the responses between naive and vehicle treated mice were observed in any of the three age groups. As shown in Table I and Fig. 4 the rank order of percent suppression of antiSRBC activity due to BaP exposure in the three age groups was 2 3 - 2 6 months > 16-18 months > 3 - 6 months. No significant differences in total spleen counts or viabflities were noted between mice of the same age group. Susceptibility to immunological suppression by BaP may therefore be due in part to the "immunological age" of the host.

338 100

j

90

1 :.:.:.,.,.:.

y~

2

......o... ,........,

80 Z O

o') UJ n" O.

I

I 4-5 MONTHS 23-26

MONTHS

..........

70

;!i!ii;iiii -

60

-

+; . . . . .

50

IX.

40

-s_ .....

+ -

+

. _ . . . ,,

30 20"

o.,...

,,.,.°,,°.,

gi

10-

._,-_-_:_:.:

°:.:-:-:.:

,-.:.:.:.-.: :.+.,..o-..-,

i:i:i:~$

0

10 BaP (p.g/rnl)

1

50

Fig. 3. Comparison of percent suppression of in vitro, anti-SRBC plaque forming responses in young and aged mice as a function of BaP concentration. Percent suppression was calculated from the results shown in Figs. 1 and 2 as follows: 1 - (test response/vehicle response) × 100. Data from 23- to 26m o n t h and 3- to 5-month-old mice were statistically compared as described in Materials and Methods at each BaP concentration. Data for each group is presented as Mean + S.E. of the mean. Significance at P < 0.01 is indicated by a double asterisk.

TABLE 1 SUMMARY OF YOUNG AND AGED MICE EXPOSED IN VIVO TO BaP

Treatment b

Age (months) a 3-6

PFC/104 viable Naive Vehicle BaP

16-18

23-26

Exp. 1

Exp. 2

Exp. 1

Exp. 2

Exp. 1

Exp. 2

spleen cells c 1776_+58 1821 +_135 1040 ± 9 9 * * (43) d

1974_+63 1878 -+81 1457 _+88* (23)

742_+70 685 -+201 257_+144" (63)

1138+331 1302 ± 2 8 5 220 ± 1 0 5 " * (84)

100_+17 92 _+26 7_+4" (93)

126_+13 133 _+27 10_+5" (93)

aEach group included 4 mice except the 23-26-month-old naive group in experiment 2 which included 3 mice due to the exclusion of 1 mouse that developed neoplasia. bMice were treated with BaP at 40 mg/kg for 4 days prior to and 4 days following immunization with SRBC. CSpleens of individual mice were analyzed foUowing immunization in vivo with SRBC. The data are presented as Mean _+S.E. PFC]106 viable spleen ceils for each group. Numbers in parentheses indicate the % suppression of the BaP group compared to the vehicle group. Groups that were statistically different from the appropriate vehicle group are indicated by a single asterisk when significance was at the P < 0.05 level and by a double asterisk when P < 0.01. dThe number in parentheses indicates the % suppression for the BaP group as compared to the corresponding veheile group. Percent suppression was calculated using the formula: 1 -- (test response/ vehicle response) × 100.

339 100' 90.

I I~

80' Z

70'

I 3-6 MONTHS 16-18 MONTHS 23-26 MONTHS

0 60' I,U ha. Q.

r.D

50' 40' 30' I

20' 10' 0 1

EXPERIMENT NUMBER

Fig. 4. Comparison of percent suppression of in vivo anti-SRBC plaque forming responses in young and aged mice exposed to BaP. Percent suppression was calcuhted from the results presented in Table I as follows: 1 - (test response/vehicle response) X 100. Data from 23-26- and 16- to 18-month-old mice were statistically compared to the 4-5-month-old group as described in Materials and Methods. Data for each group is presented as Mean _+S.E. of the mean. Significance at P < 0.05 is indicated by a single asterisk while significance at P < 0.01 is indicated by a double asterisk. Two independent experiments are presented.

DISCUSSION The above results are, to our knowledge, the first description o f the effects o f an environmental chemical on the immune system o f aged mice. Susceptibility to immunological suppression by PAHs, of which BaP is representative, has been shown in this report to be roughly proportional to chronological age from 4 months onwards. That chronological age and immunological age are interrelated has been demonstrated by numerous investigators [7,8,13,14]. Maximum immunological competency is achieved approximately 6 - 8 weeks following birth. Depending on the strain o f mouse a gradual decline in immune competency, especially as concerns T cell responsiveness, has been observed from approximately 8 months o f age onwards. BaP is a highly prevalent environmental chemical [17]. The vast majority of the approximately 900 tons o f BaP that is annually emitted into the atmosphere results from the incomplete combustion of organic matter such as coal [17]. Tobacco and marijuana smoke, as well as charcoal-broiled foods, also contain significant levels o f PAHs. The suppressive effect o f PAHs in general and BaP in particular on humoral mediated immunity has been well documented [ 1 - 3 ] although its effects on cell mediated immunity are less clear. Additionally, a large body o f information concerning the metabolism and toxicity o f PAHs has been reported [ 18,19]. As such, BaP is a highly useful compound with which to assess the risk that chemicals in the environment pose for the immune system.

340 We employed an experimental design in which mice of different ages were exposed in vivo to BaP for an acute period. This allowed the separation of possible immuntoxic

effects from the well documented mutagenic and carcinogenic effects of BaP that are the result of prolonged chronic exposure. It should be noted, however, that the assessment of exposed animals over a 3--5-month period for possible BaP induced tumor formation was not performed due to the limited availability of aged mice. Possible differences in BaP pharmacokinetics between the three age groups may account in part for the observed results. Birnbaum [20] has shown increased rates of retention and decreased tissue clearance of a hexachlorobiphenyl in senescent rats as compared to young. The data presented in Figs. 1 - 4 and Table I provide initial evidence to support the hypothesis that aged animals which are in an immunodeficient state may be more susceptible to the immunosuppressive action of BaP than young adults. The dose response curves shown in Figs. 1 and 2 demonstrate that in vitro, splenocytes from 23- to 26-month-old mice displayed significantly increased susceptibility to the immunosuppressive effects of BaP with complete suppression of antibody forming responses being achieved at 1-1.5 logs lower BaP dose than observed in splenocytes from 3- to 6-month-old mice. As has been extensively documented, the anti-SRBC plaque forming response of naive or vehicle exposed mice was shown to be inversely proportional to chronological age (Table I). Twenty-three- to 26-month-old mice which had the lowest naive and vehicle anti-SRBC response also experienced the greatest percent suppression due to BaP exposure. Three- to 6-month-old mice which displayed the highest anti-SRBC response were the least BaP suppressed of the three groups. Sixteen- to 18-month-old animals were intermediate in their degree of suppression. It is interesting to note that a recent study reported increased susceptibility of immunodeficient mice to the immunotoxic effects of environmental chemicals. Porter et al. demonstrated that the reproductive capacity and growth of marginally malnourished mice are significantly diminished when exposed to a combination of environmental chemicals and infectious agents [21]. Evidence of immunosuppression induced by environmenal chemicals, such as that reported here for BaP, does not necessarily imply that overt clinical disease is imminent. The definitive experiments needed to test this possibility have yet to be performed. Thus, it may be concluded that: (1) aged animals due to their immunodeficient state are potentially more susceptible to the immunotoxic actions of BaP than are young adults: and (2) evaluation of the health hazards posed by exposure to environmental chemicals must include immunodeficient animal models, aged or otherwise, in which possible immunotoxicity may be more fully and realistically evaluated. Current studies in our laboratory are aimed at identifying the cellular target(s) and mechanisms of BaP induced immunosuppression. REFERENCES

1 R.G. Harvey, Polyeyclic hydrocarbons and cancer.Am. Sci., 70 (1982) 386-393. 2 R.A. Malmgren, B.E. Bennison and T.W. McKinley, Reduced antibody titers in mice treated with carcinogenic and cancer chemotherapeutic agents. Proc. Soc. Exp. Biol. Med., 79 (1952) 484-488.

341 3 J.H. Dean, M.I. Luster, G.A. Boorman, L.D. Lauer, R.W. Ruebke and L. Lawson, Selective immunosuppression resulting from exposure to the carcinogenic congener of benzopyrene in B6C3F1 mice. Clin, Exp. Immunol., 52 (1983) 199-206. 4 R.E. Faith and J.A. Moore, Impairment of thymus-dependent immune fractions by exposure of the developing immune system to 2,3,7,8-tettachlorodibenzo-p-dioxin (TCDD). J. Taxicol. Environ. Health, 3 (1977)451-464. 5 M.I. Luster, R.E. Faith and G. Clark, Laboratory Studies on the immune effects of halogenated aromatics. Ann. N. Y. Acad. Sci., 320 (1979)473-486. 6 T. Kalland, Alterations of antibody response in female mice after neonatal exposure to diethylstilbestrol. J. lmmunol., 124 (1980) 194-198. 7 T. Makinodan and M.M.B. Kay, Age influence on the immune system. Adv. Immunol., 29 (1980) 287-330. 8 R.E. CaUard and A. Basten, Immune function in aged mice. IV. Loss of T and B cell function in thymus-dependent antibody response. Fur. J. Immunol., 8 (1978) 552-558. 9 F. Chino, T. Makinodan, W.E. Lever and W.J. Peterson, The immune systems of mice reared in clean and in dirty conventional animal farms: I. Life expectancy and pathology of mice with long life-spans. Z Gerontol., 26 (1971)497-507. 10 T. Makinodan, J.W. Albright, F.I. Good, C.P. Peter and M.L. Heidrick, Reduced humoral immune activity in long-lived mice: An approach to elucidating its mechanisms. Immunology, 31 (1976) 903-911. t l C.C. Stewart, Murine mononuclear phagocytes from bone marrow. In D.O. Adams, P.J. Edelson and H. Koren (eds.), Methods for Studying Mononuclear Phagocytes, Academic Press, New York, 1981, pp. 5 - 2 0 . 12 P.H. Bick and D.C. Shreffler, Selective effects of anti-la serum and complement treatment upon polyclonal B lymphocyte responses to dextran sulphate and lipopolysaccharide. J. lmmunol., 123 (1979) 2298-2303. 13 S. Kishimoto, T. Takahama and H. Mizumachi, In vitro immune response to the 2,4,6-trinitrophenyl determinant in aged C57B1/6J mice: Changes in the humoral response to, aridity for the TNP determinant and responsiveness to LPS effect with aging. J. Immunol., 116 (1976) 294-300. 14 A.A. Nordin and M.A. Buchholz, In vitro antibody response in aging animals. In W.H. Adler and A.A. Nordin (eds.), Immunological Techniques Applied to Aging Research, CRC Press, Inc., Florida, 1981, pp. 99-108. 15 A. C•utinh• and G. M•uer• B cell mit•g•nic pr•perties •f thymus-independent antigens. Nat.• New Biol., 245 (1973) 12-14. 16 B. Andersson and H. Blomgren, Evidence for thymus-independent humoral antibody production in mice against polyvinylpyrrolidone and E. coil iipopolysaceharigle. Cell. Immunol., 2 (1971) 411-424. 17 E.J. Baum, Occurrence and surveillance of polycyclic hydrocarbons. In H.V. Gelboin and P.O.P. Ts'o (eds.), Polycyclic Hydrocarbons and Cancer, Vol. 1, Academic Press, New York, 1978, pp. 45-70. 18 J.B. Vaught, H.L. Gurtoo, N.B. Parker and J.A. Hauser, Benzo[a] pyrene metabolism and macromolecular binding in strains of Ah responsive and Ah non.responsive mice. Chem..Biol. Interact., 32 (1980) 151-170. 19 M.A.Zedeck, Polycyclic aromatic hydrocarbons: A review. J. Environ, Pathok Toxicol., 3 (1980) 537-567. 20 L.S. Birnbaum, Distribution and excretion of 2,3,6,2~,3',6': ahd 2,4,5,2~,4t;5'-heXachlorobiphenyl in senescent rats. ToxicoL Appl. PharmacoL, 70 (1983) 262-272. 21 W.P. Porter, R. Hinsdill, A. Fairbrother, L.J. Olson, J, Jaeger, T. Yuill, S. Bisgaard, W.G. Hunter and K. Nolan, Toxicant-disease-environment interactions associated with suppression of immune system, growth and reproduction. Science, 224 (1984) 1014-1016.