The effect of formaldehyde exposure upon the mononuclear phagocyte system of mice

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

88,165-l 74 (1987)

The Effect of Formaldehyde Exposure upon the Mononuclear Phagocyte System of Mice’ DOLPH

0. ADAMS, T. A. HAMILTON,

L. D. LAUER, AND J. H. DEAN

Department ofPathology, Duke University, Durham, North Carolina 27710; and Department of Cell Biolog.v, Chemical Industry Institute @“Toxicology, Research Triangle Park. North Carolina 27709

Received July 15, 1986; accepted December 5, 1986 The Effect of Formaldehyde Exposure upon the Mononuclear

Phagocyte System of Mice. (I 987). Toxicol. Appl. Pharmacol. 88, 165-174. The vapors of formaldehyde have been reported to represent a potential health hazard, resulting in an increased incidence of carcinomas ofthe nasal turbinates in experimental animals. To determine the potential role of alterations in the mononuclear phagocyte system (MPS) induced by inhalation of formaldehyde, we studied the systemic effects of exposure upon macrophages. Specifically, we examined the effects of formaldehyde exposure upon development of the MPS by use of an established system of quantitative objective markers, which characterizes and classifiespopulations of murine macrophages into several developmental stages. Exposure of mice to 15 ppm of formaldehyde for 6 hr daily for 3 weeks did not alter the number or impair the function of resident peritoneal macrophages, although this exposure increased (approximately twofold) competence for release of H202 from the macrophages. Furthermore, formaldehyde exposure did not alter the tumoricidal activation or differentiation of macrophages produced by the defined stimulant MVE-2. The data thus indicate that exposure of mice to formaldehyde can induce selective systemic alterations in the function of the MPS for Hz02 production, a change which has been shown in other studies to increase the frequency of mutagenesis. 0 1987 Academic Press, Inc. ADAMS,

D. O.,

HAMILTON,

T . A.,

LAUER,

L. D.,

Aldehydes are highly irritating compounds which damage the nasal cavity and cause ulcerations, inflammation, metaplasia, and keratinization (Appelman et al., 1982; Buckley et al., 1984). Generally, unsaturated aldehydes such as formaldehyde and acrolein are more potent than saturated aldehydes such as acetaldehyde. Low-molecular-weight aldehydes are highly soluble and dissolve readily in the upper airways. Formaldehyde and acrolein have comparable acute toxicity, since effects are observed with both at about 0.4 to 6 ppm. Formaldehyde vapors, which are prevalent in the environment through numerous sources including automotive ex’ Supported in part by USPHS Grant ES 02922. 165

AND

DEAN,

J. H.

haust, industrial smoke, cigarette smoke, industrial processes and release from ureaformaldehyde resinous products, may pose potential health hazards to exposed humans (For review, see Report of the Consensus Workshop on Formaldehyde, 1984). In chronic inhalation studies in rats, formaldehyde exposure has led to an increased incidence of carcinomas of the nasal turbinates (Kerns et al., 1983) and increased cell proliferation (Swenberg et al., 1980). Additional concerns over an increased incidence of upper respiratory infections among individuals or rodents chronically exposed to formaldehyde emissions (Burdach and Wechselberg, 1980) or to various other aldehydes (Jakab, 1977; Astey and Jakab, 1983) have prompted a recent analysis of the 0041-008X/87

$3.00

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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systemic immune effects of formaldehyde exposure (Dean et al., 1984). In these studies, most immune and host resistance functions (i.e., functions involving T and B lymphocytes and macrophages) were not impaired by exposure to formaldehyde, although some enhanced functions in the mononuclear phagocyte system (MPS) were observed (Dean et al., 1984). Accordingly, an analysis in depth was undertaken of the effects of formaldehyde upon the MPS. The literature concerning the effects of various xenobiotics upon the MPS is voluminous, but difficult to place in a rational framework (Dean and Adams, 1985; Adams and Lewis, 1985; Adams et al., 1986). One reason for this confusion is that most studies have treated the MPS as a static population of cells (Dean and Adams, 1985; Adams and Lewis, 1985). while in reality the MPS is a hostwide system of leukocytes whose state of maturation is ever changing (Adams and Hamilton, 1984). The development of a panel of objective markers characterizing macrophages in each of the various stages of maturation has permitted identification of the degree of differentiation possessed by a given population of macrophages (Adams and Hamilton, 1984). Another problem is that many studies have studied macrophages taken from the site where the xenobiotic lodges or is injected. In such studies, effects of the xenobiotic may be difficult to distinguish from effects due to nonspecific local irritation and inflammation (see Adams and Lewis, 1985). Over the past several years, several xenobiotics have been found to act upon the MPS by altering development patterns, including xenobiotics which induce maturation; others which inhibit the maturation normally induced by other signals; and. some which have both effects (for reviews, see Dean and Adams, 1985; Adams and Lewis, 1985; Adams et al., 1986). Of note, these effects were observed in mononuclear phagocytes taken from sites distant from those where the xenobiotic was introduced, indicating that perturbations in the MPS can be induced systemically as well

ET

AL.

as locally. Thus, many xenobiotics act upon the MPS by systemically inducing perturbations in its development (Dean and Adams, 1985; Adams and Lewis, 1985; Adams et al.. 1986). Based on these considerations, we examined the systemic effects of formaldehyde exposure upon maturation of the MPS. Systemic effects were examined for three reasons. First, several important xenobiotics cause pertubations in the mononuclear phagocyte system as a whole, as opposed to effects confined to the site of injection (Dean et al., 1986, 1987: Murray et al., 1985). These effects, which are quite selective for the individual xenobiotic tested, imply that the changes are unique to the xenobiotic under examination and are not due to local irritation and inflammation. Second, changes in mononuclear phagocytes throughout the body are of greater potential importance to the host than effects confined to one area. Third, we wished to test the hypothesis that the previously observed systemic alteration in host resistance of Listeria monocytogenes in formaldehyde-exposed mice (Dean et a/. , 1984) was due to systemic alterations in the MPS. We examined maturation because macrophages, unlike neutrophils, are not end cells, but are long-lived cells which pass through several stages of development following release from the marrow and migration to the tissues (Adams and Hamilton, 1984). As they pass through these developmental stages, they acquire and lose many functional capacities (Adams and Hamilton, 1984). One such capacity is the ability to secrete reactive oxygen intermediates (ROI) (Nathan and Root, 1977; Murray, 1984). Resident murine peritoneal macrophages from the unmanipulated peritoneal cavity secrete very low amounts of H202, whereas cells which have been exposed to an irritant. an inflammatory signal, or mycobacteria are able to secrete copious quantities of H202 (Nathan and Root, 1977; Murray, 1984). We report here that systemic exposure to formaldehyde, an irritant, carcinogen, and com-

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pound of significant human exposure, causes a substantial increase in the ability of elicited macrophages to secrete H202. METHODS iinimals. Female specihc-pathogen-free (SPF) B6C3Fl (C57B1/6 X C3H) mice 6 to 8 weeks of age, weighing 1822 g were obtained through Charles River (Portage, MI). All animals were quarantined upon arrival for 15 days in a mass air displacement room (Bioclean, Hazleton Systems, Inc., Vienna, VA) and housed five per cage in hanging stainless-steel mesh cages (Hazleton Systems Inc.). Animals received a defined laboratory chow (NIH-07, Ziegler. Bros.. Gardner. PA) and purified tap water ad lihiturn. Randomly selected animals were necropsied on arrival and were found to be free ofknown rodent pathogens (standard screening necropsy, Microbiological Associates, Bethesda, MD). Sentinel animals, bled at weekly intervals throughout the study, were also free of virus titers. E.upo.rures. The exposures were conducted in an 8-m3 stainless-steel and glass chamber as previously described (Dean PI al.. 1984). The HCHO gas was generated by thermal depolymerization ofparaformaldehyde (Aldrich Chemical), a solid of -95% formaldehyde. The paraformaidehyde was contained in a stainless-steel canister enclosed within a thermally controlled oven. Air passing through the canister at a rate of 500 to 1000 ml/min carried the HCHO gas through heated stainless-steel tubing to the air supply duct of the chamber. Chamber concentrations were monitored continually with an infrared spectrophotometer (Miran 1A. Wilkes Scientific) at a wavelength of 3.58 pm and a pathlength of 20.25 cm. The infrared analyzer was calibrated with a paraformaldehyde permeation tube (VlCl Metronics) whose permeation rate was quantitated by a chromotropic acid colorimetric method (Katz, 1977). Chamber air flow, temperature. and relative humidity were monitored and recorded hourly. Mice were exposed to - 15 ppm of HCHO for 6 hrfday, 5 days/week for 3 weeks. Three groups of mice were exposed. The actual HCHO exposures. for the three groups of mice exposed to HCHO, were 15.02 + 0.1 I. 14.77 f 0.34, and 14.78 f 0.20 ppm, respectively; these values represent the mean HCHO concentrations &SE for the 2 1-day exposure period. Agematched control mice received ambient air. This concentration of formaldehyde was selected because of previous studies showing enhanced resistance to L. monocyto,reenesand because this dose approaches the maximum tolerated dose of HCHO in mice (Dean et al., 1984). We elected this duration and concentration of HCHO exposure based on these previously published studies in which enhanced resistance to L. monocytogenes was observed following exposure of mice to this concentration of

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167

HCHO for this period of time. The present conditions were thus chosen to relate to previous findings which implicate the mononuclear phagocyte system (the object of study in these studies) and also to examine the highest possible tolerated doses. Recovery ufmacrophuges. Peritoneal leukocytes were collected by peritoneal lavage from control and HCHOexposed mice 2-3 days after the final exposure, using 10 ml of calcium- and magnesium-free Hanks’ buffered salt solution (HBSS) containing 10 units of heparin/ml. Peritoneal macrophages (M@) elicited by pyran copolymer (MVE-2, agift from Adria Laboratories, Columbus, OH) were obtained 6 days after intraperitoneal injection of 200 pg of MVE-2 to both treatment and control groups. These groups were included to determine whether HCHO exposure altered M# elicitation or activation by the prototype activator, MVE-2 (Adams et al.. 1983; Dean et a/., 1978). Cells from several animals were pooled to yield sufficient numbers of macrophages to permit performance ofall assaysofmacrophage function on the same group of macrophages. Cell and differential counts were both performed in triplicate. The cells were placed in microexudate-coated tissue culture flasks (Costar, Cambridge. MA) and incubated 2 hr for adherence purification at 37°C in a humidified 5% CO?-air atmosphere. Nonadherent cells were decanted and the macrophage-rich adherent cells were washed thrice with 10 ml of HBSS. This procedure resulted in monolayers of >95% pure macrophages, asdetermined by morphology, phagocytosis, and staining for nonspecific esterase (Johnson et al.. 1983). In some experiments, the adherent cells were then gently scraped with a cell scraper and cell counts were performed. The cultures were adjusted to contain equal numbers of adherent macrophages, whether the macrophages were derived from experimental or control mice. This point was quantified by use of inverted phase microscope as previously described (Johnson et al.. 1983) or cell counts. Quuntificution of mucrophagejiinctions. Macrophage phagocytosis was determined using “Cr-labeled sheep red blood cells (SRBC) as described previously (Johnson et al.. 1983). Binding and lysis of P8 15 target cells to macrophage monolayers were also quantified as previously described (Johnson ef al.. 1983). HzOz production was measured in resident and elicited macrophages in both control and HCHO-exposed groups. H202 was measured after phorbol myristate acetate (PMA) was added in nanomolar quantities to the in vitro cultures using a colorimetric method previously described in detail (Chen et al., 1982). The assayis linear over the time of release and gives data quantitatively similar to those obtained using the scopoletin assay (Cohen et al., 1982). Release was measured in the absence and presence of PMA. Macrophage enzymes. Peritoneal cells were collected from exposed animals and the cell counts adjusted to 30 X 106/ml. The erythrocytes (RBC) were lysed by an ammonium chloride Iysing buffer and the cells solubilized

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with 0.05% T&on-X 100. The ectoenzyme leucine aminopeptidase (LAP) was quantified as previously described in detail (Morahan et al., 1980). The LAP method (Bio-Dynamics, Indianapolis, IN) is based upon the enzymatic hydrolysis of L-leucine-p-nitroanilide by LAP to p-nitroanihne which is chromogenic at 400 nm. This enzyme has been described to distinguish inflammatory from activated macrophages (Morahan et al.. 1980). Assays were run so that nonomoles of substrate hydrolyzed per minute per milligram of protein were quantified. The data are reported in units where 1 unit equals 1 nanomole hydrolyzed per minute per mg of macrophage protein. The amount ofacid phosphatase in the macrophages was quantified as previously described (Johnson et al., 1983). The protein content of the macrophage monolayers was determined by the method of Bradford as previously described (Johnson et al.. 1983). Activation of macrophages in vitro. Macrophage monolayers (3 X IO5cm2) from mice given peptone broth were treated with macrophage-activating factor (MAF) and lipopolysaccharide (LPS) as described in detail (Johnson et al., 1983). Cytolysis of P8 I5 tumor cells was then quantified (Johnson et al., 1983). Statistical analysis. Statistical difference between responses in MI#I from control and treated mice were determined by Dunnett’s multicomparison t test.

RESULTS Eflects on Number Macrophages

of Resident and Elicited

Exposure of mice to a formaldehyde concentration (i.e., to - 15 ppm), which may be carcinogenic upon prolonged exposure, for 6 hr a day for 5 days a week for 3 weeks did not appreciably alter the number of resident macrophages in the peritoneal cavity or that elicited in response to MVE-2 (Table 1). Furthermore this level of exposure did not induce generalized toxicity as indicated by any differences in body or lymphoid organ weights between exposed and control groups (Dean et al., 1984). Effects on Macrophage Development Xenobiotics have been previously observed to affect the MPS in two general ways: (a) induction of macrophage maturation or (b) inhibition of development in macro-

ET AL. TABLE 1 EFFECTOFFORMALDEHYDEONNUMBEROFRESIDENT ANDELICITEDPJZRITONEALM$“

Number of Mob Control mice Resident M@J MVE-2 Elicited

4.2 f 0.2 10.8 f 0.7

Formaldehydetreated mice 5.4 f 0.3 12.0 IL 0.4

’ Representative experiment, similar results were obtained in two additional experiments. b The data shown are the .rk SE of peritoneal macrophages obtained from the peritoneal cavity of 10 mice per group and are reported as $/mouse x 1Om6.

phages which are maturing in response to a defined stimulant (Dean and Adams, 1985; Adams and Lewis, 1985). To determine if the first possibility occurs following HCHO exposure, resident populations of peritoneal macrophages were examined by using a panel of objective quantitative markers that discriminate between populations of macrophages in the various stages of development (Johnson et al., 1983). Overall, expression of these markers by resident macrophages from control or formaldehydeexposed mice indicated that both populations of macrophages were in the basal or resident stage of development (Table 2). These observations were confirmed when we tested competence of the macrophages to bind tumor cells (Fig. 1A). or to lyse tumor cells (Fig. 2A). The macrophages from HCHO-exposed mice did, however, have a lower content of LAP (Table 2). These data alone would raise the possibility that formaldehyde had enhanced the maturation of the resident peritoneal macrophages. Since we did not observe other evidence for enhanced maturation in the competence of these cells to phagocytosed antibody-coated erythrocytes, for example, produce more acid phosphatase, secrete more hydrogen peroxide, bind more tumor cells, or lyse more tumor cells, the data overall do not suggest that the resident macrophages were

FORMALDEHYDE

AND

TABLE

2

EFFECT OF FORMALDEHYDE EXPOSURE ON PHAGOCYTIC AMINOPEPTIDASE, AND CONTENT OF ACID PHOSPHATASE Resident

Control Phagocytosish of ElgG Content of LAP’ Content of acid phosphatased

M$

169

M@

FUNCTION, THE ECTOENZYME LEUCINE IN RESIDENT AND MVE-~-ELICITED M@” MVE-2-Elicited

from Formaldehydeexposed mice

mice

1.55 io.l 81.7 + I 843 f 80

Control mice

1.60 f 0.2 24.5 + 2 629 ? 32

Formaldehydeexposed mice

3.25 f 0.3 41.4% 5 1984 + 101

’ Similar data were obtained in two additional experiments. All data are given as .\-i SE. ’ Data are given as cpm of ingested Naz5’Cr0,-labeled EIgG per 3 X lo5 Mb. ’ Content of LAP is given as units (as defined under Methods) per milligram of M$I protein. ‘Content of acid phosphatase is defined as nanomoles of substrate hydrolyzed per minute protein.

clearly pushed to a further stage of development. Thus, formaldehyde exposure did not induce significant systemic maturation of resident tissue macrophages. To test the second possibility, we examined the maturation of macrophages induced to

I

I 1.0

1 2.0 NUMBER

1 3.0

1 1.0

I 2.0

I 3.0

OF MP) x IO-5

FIG. 1. Effect of formaldehyde exposure on selective binding of tumor cells by resident and primed peritoneal macrophages. Resident peritoneal macrophages were obtained from unmanipulated mice (A) and primed macrophages were obtained with MVE-treated mice (B). The two types of macrophages were obtained from untreated mice (-) and mice which had been exposed to formaldehyde (---). The cells were plated to yield comparable numbers of adherent purified mononuclear phagocytes as described under Methods. The number of P8 15 tumor cells selectively bound by the macrophages was then quantified as described under Methods. The data shown represent the .c + SE of triplicate determinations. These results were obtained in two separate experiments.

MI#J from

3.30 * 0.2 40.2 f 3 1931 Ik 120

per milligram

of Mb

the primed stage of development by a chemically defined stimulant-MVE-2 (Adams et al., 1983). These macrophages differed significantly from the resident macrophages in increased competence for phagocytosis of IgG-coated red cells, in chemical content of

/ , 1.0

2.0 NUMBER

3.0

1.0

2.0

3.0

OF PAP) x 10-S

FIG. 2. Effect of formaldehyde exposure on lysis of tumor cells by resident and primed peritoneal macrophages. Resident peritoneal macrophages were obtained from unmanipulated mice (A) and primed macrophages were obtained with MVE-treated mice(B). The two types of macrophages were obtained from untreated mice (--) and mice which had been exposed to formaldehyde (---). The cells were plated to yield comparable numbers of adherent purified mononuclear phagocytes as described under Methods. The net cytolysis of P8 I5 tumor cells in the presence of LPS by the macrophages was then quantified as described under Methods. The data shown represent the ? f SE of triplicate determinations. These results were obtained in two separate experiments.

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the lysosomal hydrolase acid phosphatase (AP), and in decreased activation of leucine aminopeptidase (Table 2). This development was not altered in the formaldehyde-exposed mice (Table 2). The pyran (MVE-2)-primed macrophages, as previously reported, bound tumor cells extensively (Fig. 1B) and lysed them efficiently in the presence of small amounts of endotoxin (i.e., 10 rig/ml) (Fig. 2B). These two acquired functions were not significantly altered in the macrophages from formaldehyde-treated mice (Figs. 1 and 2). Cytolysis by the primed macrophages in the presence of endotoxin was slightly higher at the concentration of 2 X lo5 adherent macrophages (i.e., 28% vs 37% cytolysis), but this difference was not judged to be biologically significant, since lysis at the other two concentrations of macrophages was comparable. Neither population of primed macrophages lysed the tumor target cells in the absence of endotoxin (Fig. 2B). Thus, formaldehyde exposure neither inhibited the development of primed macrophages induced by MVE-2 nor pushed these macrophages further to the fully activated state. Efects on Macrophage Competence to Release Reactive Oxygen Intermediates WI) Competence to release ROI extensively is an acquired property of macrophages, which develops along with maturation for lytic competence but which is independently regulated (Adams and Hamilton, 1984). Resident peritoneal macrophages did not release detectable amounts of H202 natively or in response to the pharmacologic stimulus PMA (Table 3). The primed macrophages elicited by MVE-2 had acquired competence for pharmacologic release of Hz02 ; this was significantly increased (i.e., - twofold) in macrophages from formaldehyde-exposed mice (Table 3). This observation indicates that exposure to formaldehyde can, on a systemic basis, significantly enhance macrophage competence to release ROI.

ET AL. TABLE

3

EFFECTOFFORMALDEHYDEEXPOSUREON MACROPHAGECOMPETENCETORELEASE HzOL’ Released

Mc$ from control mice Resident M$ Nil PMA MVE-2 Elicited Nil PMA

H20z * M$ from formaldehydeexposed mice

0 0

0 0

0 42.5 f 3.3

0 78.2 f 1.7’

M$J

g Similar data were obtained in two additional experiments. ’ Data are given as nanomoles of Hz02 released per hour per milligram of M+ protein in response to no stimulant or PMA as described under Methods. c Significantly different from control macrophages at p < 0.001.

DISCUSSION The mononuclear phagocyte system represents a major and important target for environmental pollutants for a variety of reasons (for reviews, see Adams and Lewis, 1985; Adams et al., 1986; Dean and Adams, 1985). It is well established that mononuclear phagocytes are capable, at the site of inhalation, deposition, injection, or exposure in causing local changes in the population of inflammatory leukocytes responding to the xenobiotic in question. Such changes may be attributable in part to nonspecific local inflammatory effects of the xenobiotic and may well be of particular relevance only to the site of deposition of the xenobiotic. Systemic effects of mononuclear phagocytes. by contrast, are of more potential significance because they imply that the mononuclear phagocyte system of the entire host is perturbed so that potential deleterious effects can be seen in sites distant from where the xenbiotic in question has actually lodged. Over the past several years, we have examined this possibility and found

FORMALDEHYDE

that three xenobiotics of considerable environmental concern (i.e., phorboi diesters, diethylstilbesterol, and dimethylbenzanthracene) all cause systemic pertubations in the mononuclear phagocyte system (Dean et al., 1986, 1987; Murray et al., 1985). These effects have been determined by examining mononuclear phagocytes distant from where the xenobiotic of interest is lodged. For these studies, we routinely employed peritoneal macrophages as a readily obtainable, stable population, which will permit detailed comparisons of changes in the macrophages from xenobiotic to xenobiotic. Furthermore, the complex effects on the MPS of these three xenobiotics have been distinctly different, implying that the systemic changes are unique to the xenobiotic in question and are not simply the result of local irritation and inflammation (Adams et al., 1986). The data presented here support two major conclusions: (a) formaldehyde exposure does not appear to perturb systemic development of the MPS in regard to activation for tumoritidal function; and (b) such exposure does enhance development for release of reactive oxygen intermediates (Table 3). One cause for this systemic alteration may be the local inflammation produced in the upper airway of these animals by this irritant gas. This disparity in effects on two different forms of activation is not surprising. In general, macrophages develop competence for performance of different complex functions, each of which exhibits different physiologic requirements and each of which is differentially regulated (Adams and Hamilton, 1984). The present observations support the growing body of evidence indicating that many xenobiotics of importance act upon the MPS and do so by perturbing the delicate balance of inductive and suppressive signals that control the dynamic state of development of this hostwide cell system (Dean and Adams, 1985; Adams and Lewis, 1985). Previous studies have shown that exposure of mice to formaldehyde resulted in enhanced systemic resistance to L.. monocyto-

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genes (Dean et al., 1984), although exposure of mice to the unsaturated three-carbon aldehyde, acrolein, has been shown to suppress intrapulmonary killing of Proteus mirabilis (Jakab, 1977). Suppressed host resistance was similarly obtained when mice were exposed to 3 or 6 ppm acrolein for 8 hr and then infected with Staphylococcus aureus (Astry and Jakab, 1983). The mechanisms by which an unsaturated aldehyde such as acrolein may impair bacterial defenses is unknown but are suggested to involve suppression of phagocytosis by alveolar macrophages (Green et al., 197 1). Based on similarities between HCHO and acrolein in structure and biological effects, it was suspected that formaldehyde might alter susceptibility to bacterial challenge and possibly impair macrophage microbicidal function. The studies described in this report suggest the contrary, that physiological activation of macrophages was not impaired by inhalation exposure to formaldehyde. Increasing evidence implicates free radicals derived from reactive oxygen intermediates in human diseases including cancer, lung disease, kidney disease, and the general process of aging (Ames, 1983). A potential source of abundant ROI is inflammatory leukocytesmacrophages and neutrophils-which release H202 and 02-copiously when appropriately stimulated (Nathan and Root, 1977; Goldstein et al., 198 1). Since most tumor promoters active in the two-stage mouse skin model are both inflammatory agents (i.e., cause an influx of inflammatory cells) and potent stimulants of the respiratory burst in macrophages and neutrophils, it has been hypothesized that tumor promoters (e.g., PMA) act in vivo in part by attracting inflammatory cells and stimulating them to secrete ROI which causes oxidative DNA damage in surrounding cells. Human peripheral blood leukocytes, when stimulated with TPA, suffer a high degree of single-strand breaks in their DNA (Bimboim, 1982). These leukocytes can induce mutagenesis in bacteria and sister-chromatid exchanges in cocul-

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tured mammalian cells (Weitberg et al., 1983; Weitzman and Stossel, 1982). When mitogen-induced lymphocytes are stimulated with TPA, gross chromosome breakages and alterations occur (Weitberg et al., 1983). In fact, macrophages from within tumor sites are mutagenic (Fulton et al., 1984). We have recently found that inflammatory macrophages stimulated with TPA induce 5,6-ring-saturated thymine bases in the DNA of cocultured mammalian cells (Lewis and Adams, 1985), an alteration induced by oxygen radicals (Hariharan and Cerutti, 1974). Of equal importance, all of the above effects can be inhibited by catalase, superoxide dismutase, hydroxyl radical scavengers, or inhibitors of lipid peroxidation reactions occurring during the metabolism of arachidonic acid (Ames, 1983). Recently, Kensler et al. (1983) observed that CuDips, a compound which crosses cell membranes and has superoxide dismutase activity, inhibits promotion of tumors in mouse skin by TPA. Further evidence for the participation of ROI in carcinogenesis comes from the observation that benzoyl peroxide, an oxygen-radical-generating compound, can promote tumors in the mouse skin model (Slaga et al., 198 1). In fact, H202 is itself a weak promoter (Klein-Szanto and Slaga, 1982). Studies in vitro using enzymatically generated ROI have shown that they can initiate and promote cell transformation (Zimmerman and Cerutti, 1984). Other studies have shown that antioxidants inhibit promotion of transformation by TPA when there is no external source of ROI (Kennedy et al., 1984). Taken together, these studies indicate that ROI from exogenous or endogenous sources may be involved in carcinogenesis and cell damage. The impact of a change in the potential of the MPS to release ROI upon the host may be two-edged-that is, both beneficial and detrimental. On one hand, we have previously documented that such exposure to formaldehyde enhances host resistance to challenge with L. monocytogenes (Dean et al., 1984), an observation not surprising in

ET AL.

view of the strong correlation between macrophage activation for destruction of numerous facultative or obligate intracellular microbes and macrophage competence for release of ROI (Murray, 1984). On the other hand, we have recently observed that macrophages can induce genotoxic damage of significant mutagenic potential in bystander eukaryotic cells and that released ROI from the macrophages, at amounts comparable to those observed here, play a major part in inducing such damage (Lewis and Adams, 1985). Although the full ramifications of formaldehyde exposure upon the host remains to be defined, it does appear that the ability of one major function, release of ROI, in response to external challenges is markedly altered. The role of altered macrophages is producing the lesions observed after formaldehyde exposure (Kerns et al., 1983; Swenberg et al., 1980; Chang et al., 1983) appears to require further investigaton.

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2,283-318. ADAMS, D. O., JOHNSON, W. J., MARINO, P. A., AND DEAN, J. H. (1983). Pyran copolymer inducesincomplete activation of murine macrophage.CancerRes. 43,3633. ADAMS, D. O., AND LEWIS, J. G. (1985). The mononuclear phagocytesystemand its interactions with xenobiotics. In Immunotoxicology and ImmunopharmacoIogy(J. H. Dean, M. I. Luster, A. E. Munson, and H. Amos, Eds.), pp. 23-44. Raven Press, New York. ADAMS, D. 0.. LEWIS, J. G., AND DEAN. J. H. (1986). Activation of mononuclear phagocytes by xenobiotics of environmental concern: Analysis and host effects. In: Target Organ Toxicology: Lung(J. D. Crapo, D. E. Gardner, and E. J. Masaro, Eds.). Raven Press, New York. AMES, B. N. (1983). Dietary carcinogens and anticarcinogens. Science221,1256-1264. APPELMAN, L. M., WOUTERSEN, R. A.. AND FERON, V. J. (1982). Inhalation toxicity of acetaldehyde in rats. I. Acute and subacute studies. Toxicology 23, 293-307.

FORMALDEHYDE ASTRY, C. L., AND JAKAB, G. J. (1983). Effects of acrolein exposure on pulmonary antibacterial defenses. Toxicol. Appl. Pharmacol. 67,49-54. BARROW, C. S., AND STEINHAGEN, W. H. (1982). Design, construction and operation of a simple inhalation exposure system.Fund. Appl. Toxicol. 2,33-31. BIRNBOIM, H. C. (1982), DNA strand breakage in human leukocytes exposed to a tumor promoter, phorbol myristate acetate. Science 215, 1247-1249. BUCKLEY, L. A., JIANG, X. Z., JAMES, R. A., MORGAN, K. T., AND BARROW, C. S. (1984). Respiratory tract lesions induced by sensory irritants at the RD50 concentration. Toxicol. App. Pharmacol. 74,4 17-429. BURDACH. S. T., AND WECHSELBERG. K. (1980). Damage to health at school: Complaints due to the use of materials which emit formaldehyde in school buildings. Fortschr. Med. 98,379-384. CHANG. J. C. F., GROSS, E. A., SWENBERG.J. A., AND BARROW, C. S. (1983). Nasal cavity deposition, histopathology and cell proliferation after single or repeated formaldehyde exposures in B6C3Fl mice and F-344 rats. Toxicol. Appl. Pharmacol. 68, 16 1- 167. COHEN, M. S., TAFFET, S. M., AND ADAMS, D. 0. (1982). The relationship between secretion of HzOz and completion oftumor cytotoxicity by BCG-elicited macrophages. J. Immunol. 128, 178 l-17 185. DALHAMN, T., AND ROSENGREN, A. (197 1). Effect of different aldehydes on tracheal mucosa. Arch. Otolaryng. 93,496-500. DEAN, J. H.. AND ADAMS, D. 0. (1985). The effects of environmental agents on cells of the mononuclear phagocyte system. In The Reticula-Endothelial System: A Comprehensive Treatise, Vol. VIII, Immunopharmacology of the Reticula-Endothelial S.vstem(J. Hadden and A. Szentivany. Eds.), pp. 389-409. Plenum, New York. DEAN, J. H.. LAUER, L. D., HOUSE, R. V., MURRAY, M. J.. STILLMAN, W. S., IRONS, R. W., STEINHAGEN, W. H., BARROW, C. S., AND ADAMS, D. 0. (1984). Effect of formaldehyde exposure on immune function and host resistance in B6/C3F mice. Toxicol. Appl. Pharmacol. 72,5 19-529. DEAN. J. H., LAUER, L. D., MURRAY, M. J.. LUSTER, M. I.. NEPTUN, D., AND ADAMS, D. 0. (1986). Functions of mononuclear phagocytes in mice exposed to diethylstilbestrol: A model ofaberrant macrophage development. Cell. Immunol. 102,3 15-322. DEAN, J. H., PADARATHSINGH, M. L., AND KEYS, L. (1978). Response of murine leukemia to combined BCNU-maleic anhydride-vinyl ether (MVE) adjuvant therapy and correlation with macrophage activation by MVE in the in vitro growth inhibition assay. Cancer Treat. Rep. 62,1807-l 8 16. DEAN, J. H., WARD, E. C., MURRAY, M. J., LAUER, L. D.. HOUSE, R. B., STILLMAN, W., HAMILTON, T. A., AND ADAMS, D. 0. (1986). Immunosuppres-

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