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Fluorocarbon-enhanced mutagenesis of polyaromatic hydrocarbons
ENVIRONMENTAL RESEARCH 45, 101-107 (1988)
Fluorocarbon-Enhanced Mutagenesis of Polyaromatic Hyd rocarbons R. G. MAHURIN AND R. L. BERNSTEIN Department of Biological Sciences, San Francisco State University, 1600 Holloway Avenue, San Francisco, California 94132 Received February 24, 1987 The widely used fluorocarbon refrigerant and cleaning solvent 1,1,2-trichloro-l,2,2-trifluoroethane (Freon TF), 1 though generally considered biologically inert, enhances the metabolic activation of chemical carcinogens. Liver microsomal extracts from mice given single intraperitoneal injections of this fluorocarbon showed significant increases in their ability to activate carcinogenic polyaromatic hydrocarbons to form mutagens, compared to control mice injected with saline. Polyaromatic hydrocarbons aminofluorene and acetylaminofluorene were activated in this way. Mutagenicity was measured by a microbial assay. Both commercial grade and redistilled fluorocarbons gave similar results, that is, more highly active liver extracts after administration of the fluorocarbon preparation to mice. Neither industrial grade nor redistilled preparation was itself mutagenic. A combined liver microsomal extract from mice breathing Freon TF at 20,000 ppm in air for 8 hr also had enhanced ability to activate aminofluorene as a mutagen. Exposing mice to Freon TF by inhalation more closely matches the normal route of human exposure to fluorocarbons. The results of this study imply that low-molecular-weight fluorocarbons may pose a carcinogenic risk by acting as cocarcinogenic enhancers of carcinogen activation. The possibility that fluorocarbons are cocarcinogens in this way has apparently not been heretofore considered. © 1988AcademicPress, Inc.
INTRODUCTION Many xenobiotics, often synthetic and toxic chemical substances which are not normally associated with biological organisms, play several roles in initial carcinogenic events. Some of them are frank carcinogens ("initiators"), probably because they are mutagens or are easily activated to become mutagens by mammalian metabolism. Benzo[a]pyrene and acetylaminofluorene are examples of frank xenobiotic carcinogens. Mixed-function oxidases and other enzyme systems are responsible for the metabolism of some xenobiotics which activates them to become carcinogens (Magee, 1974). Some xenobiotics may be cocarcinogens, or substances that are not carcinogenic themselves but act synergistically with frank carcinogens to cause cancer. Two general modes of cocarcinogenesis may be stated. Cocarcinogenesis by "promotion" is one mode, which may involve the stimulation of cell growth after "initiation" or the initial mutagenic lesion has occurred. Presumably croton oil (phorbol esters), saccharin, and some hormones like diethylstilbestrol (DES) are examples of cocarcinogenic promoters. Polychlorinated biphenyls (PCBs), 3-
t Freon TF is a trademark of E. I. DuPont de Nemours & Co. 101 0013-9351/88 $3.00 Copyright© 1988by AcademicPress, Inc. All rightsof reproductionin any formreserved.
102
MAHURIN AND BERNSTEIN
methylcholanthrene, and chlorinated insecticides like DDT and lindane may have another mode of cocarcinogenicity. These latter xenobiotics have been shown to increase the activity of the microsomal enzymes responsible for the generation of other carcinogens by endogenous metabolism (Ryan et al,, 1979, Bernstein et al., 1979). Although low-molecular-weight fluorocarbons have been determined to be relatively harmless by numerous short-term tests over the past 30 years, they have some characteristics that are similar to those of known cocarcinogens. For example, they are synthetic, halogenated, and relatively unreactive chemically. They are also extremely nonpolar. In the liver and other tissues, the cytochrome P-450 system and mixed-function oxidases typically oxidize, hydroxylate, and conjugate many nonpolar compounds to aid in their solubilization and excretion. Many nonpolar compounds (including saturated fats) stimulate the activity of the microsomal oxidizing system (Conaway et al., 1977). Currently in the United States and other industrialized countries large quantities of low-molecular-weight fluorocarbons are produced and used in a wide range of products and processes ranging from refrigerants to washing of silicon chips. Some fluorocarbons ("perfluorochemical emulsions") have been used as blood substitutes (Maugh, 1979). Fluorocarbons are a well-known threat to the atmospheric ozone layer (Turio, 1985). Some anesthetic fluorocarbon gases have been implicated as carcinogens and teratogens (Corbett, 1977), though they are apparently not mutagenic (Waskell, 1978). Another hazard, unsuspected until now, exists if the low-molecular-weight fluorocarbons in widespread use are also significantly cocarcinogenic. One mechanism by which fluorocarbons may be hazardous in this way is their enhancement of enzymatic activation of carcinogenic chemicals. The present paper provides evidence for the fluorocarbon-enhanced activation of polyaromatic hydrocarbons as mutagens. METHODS Bacterial strain Salmonella typhimurium LT-2 TA98 responds well to induced mutation by polyaromatic hydrocarbon mutagens which cause frameshifts (McCann et al., 1975). Tester strain TA100 is isogenic with TA98 except that it is a sensitive indicator of substitution mutations. These bacteria are histidine auxotrophs but revert at a low rate to histidine prototrophy and grow as revertant colonies on minimal medium lacking histidine. In our experiments premutagens aminofluorene and acetylaminofluorene were incorporated into the soft agar overlay during incubation, as described by Ames et al. (1975). These compounds are converted to active mutagens by an extract (called $9) of mammalian liver which is also added to the agar overlay. The bacterial tester strains when exposed to mutagens give increased colony counts as an indicator of active mutagen produced. Microsomal $9 extracts from mammalian liver (here mouse liver) are included in the assay. The microsomal
FLUOROCARBON-ENHANCED MUTAGENESIS
103
enzymatic activities in $9 form highly active mutagenic metabolites of carcinogenic chemicals like acetylaminofluorene and benzo[a]pyrene, which would otherwise not be significantly mutagenic. In the studies reported here mutagenicity of activated forms of aminofluorene and acetylaminofluorene is measured with tester strain TA98. Weanling male Swiss Webster mice (Simonsen Laboratories) received 0.58 or 2.9 g Freon TF1/kg body wt by intraperitoneal injection. Control animals received 0.10 ml of sterile 0.85% NaC1 instead of Freon. Freon TF cleaning solvent (1,1,2trichloro-l,2,2-trifluoroethane) was obtained from DuPont de Nemours & Co. Crude Freon TF is a clear yellowish liquid which boils at 47-49°C. We redistilled Freon TF in a hot water bath with water-cooled condenser, discarding the first and last 10th fractions of distillate. RedistiUed Freon TF is colorless and boils at 48-49 °. Seven days after a single injection of either crude or redistilled Freon TF, livers were removed aseptically. Microsomal $9 extracts were prepared, stored, and used as described (Ames et al., 1975). The same volume, 0.5 ml, of $9 mix containing $9 extract and cofactors was added to each plate in each experiment. The fraction of $9 in $9 mix was held constant at 10%, except as indicated in Fig. 2. Total protein concentrations were determined by the method of Lowry et al. (1951). An additional two groups of five mice each were used to study the effects of inhaling Freon TF. One group breathed crude Freon TF at 20,000 ppm by volume in air for 1 to 2-hr periods, in an enclosed transparent gastight box of volume approx. 5 liters. Total exposure was 8 hr. One group was treated identically but without Freon. Livers were extracted and $9 was prepared as above. The activity of the combined liver extracts of each group of mice toward activating aminofluorene as a mutagen was then tested in a dose-response experiment as described above. RESULTS
No significant difference in body weight or liver weight occurred after 7 days (between controls and Freon-injected animals) as a result of single intraperitoneal injections of Freon TE The fluorocarbon is not itself mutagenic when tested against Salmonella bacterial strains TA98 and TA100 with or without $9 microsomal extract (data not shown here; results and further references are presented in detail in Mahurin, 1980). Freon TF does, however, have an effect on mutagenesis by carcinogenic aromatic hydrocarbons, as shown in Figs. 1 and 2 for tester strain TA98. Liver extracts from mice given a single intraperitoneal injection of industrial grade Freon TF are found to be considerably more active than liver extracts from mice receiving sterile saline, as seen in Fig. 1. Redistilled Freon TF gave similar results in enhancing liver enzymatic activation of carcinogenic chemicals to mutagenic forms (Mahurin, 1980). The protein concentration of $9 liver extracts from mice receiving Freon TF is considerably less than that of controls (Table 1), which means that less $9 protein,
104
MAHURIN AND BERNSTEIN I
J ¢0 O
x },z ,< I.-. cc ILl > IJJ cc
/
1 2 #g
4
/
8
16
2-ACETYLAMINOFLUORINE
FIG. l. Mutagen activation by liver extracts from Freon-injected mice: dose-response with acetylaminofluorene. Intraperitoneal injection of industrial grade Freon TF: 0.58 g/kg body wt (A) or 2.9 g/kg body weight (I). Control mice injected with 0.10 ml sterile 0.85% saline (O). Each data point is the average of three plate counts.
not more, is present in plates containing $9 from Freon-treated mice, yet the extracts are more capable of activating carcinogens. Varying the amount of carcinogen with constant microsomal protein (Fig. I), or varying microsomat protein with a constant amount of carcinogen (Fig. 2), had similar effects. In all cases liver extracts from mice injected with Freon TF were more active than extracts from mice injected with sterile saline. The number of revertants using Freon-activated mouse liver $9 reached values similar to those we obtained using Aroclor 1254-activated rat liver, namely 2000-3000 revertants per plate at doses of 8-12 ~g acetylaminofluorene or 600-800 p~g aminofluorene per plate (Mahurin, 1980). Liver extracts from mice exposed to 20,000 ppm Freon TF by volume in air had increased ability to generate active mutagens when compared to mice breathing air only (Fig. 3). We found no significant change in total body weight or weight of lungs or livers from Freon exposure by inhalation. The data of Fig. 3 indicate that the activation of small amounts of aminofluorene for bacterial mutagenesis is especially greatly enhanced by $9 extract of mice breathing Freon TE DISCUSSION
The American Conference of Governmental Industrial Hygienists has assigned a safety threshold limit for inhalation of 1000 ppm by volume for Freon TF in air for a time-weighted average workday. This is the highest limit allowable for any chemical under the current guidelines, and it implies that Freons are relatively
FLUOROCARBON-ENHANCED MUTAGENESIS
41
A
7 jj~.
105
B
6
O9 O
5
X (D t'Z < t,¢r" LLI > LLI or
CO O 4
X I.,Z< I--
3
W
>
2
W rr'
i
0.50
1.00
1.50
0J50
1.00
1.~50
MG PROTEIN PER PLATE MG PROTEIN PER PLATE
FIG. 2. Mutagen activation by varying amounts of mouse liver $9 extract. Intraperitoneal injection of industrial grade (A) or redistilled (B) Freon TF: symbols as in Fig. 1. All plates contained 600 ~g 2-aminofluorene. Each data point is the average of three plate counts.
safe. However, Vainio et al. (1980) found Freon TF to be a hepatotoxin in rats. Changes in smooth endoplasmic reticulum accompanied alterations in microsomal enzyme activities. They also found cytochrome P-450 binds Freon TE In our study mice given intraperitoneal injections of Freon TF also showed characteristically altered livers. Exposure to haloalkanes is known to cause cardiac malfunctions in animals at high doses (Back and van Stee, 1977). Reinhardt et al. (1972) discovered that trichlorotrifluoroethane (Freon 113) causes cardiac sensitization in mammals at concentrations of 5000 ppm by volume in air. Savolainen and Pf~iffli (1980) noted TABLE l PROTEIN CONCENTRATIONS IN LIVER $9 MICROSOMAL EXTRACTS
Mice injected with
No. of animals
Average protein in extract
Commercial grade Freon TF Control without Freon 0.58 g/kg Body wt 2.9 g/kg Body wt
12 12 8
14.0 mg/ml 8.6 mg/ml 7.5 mg/ml
5 6 6
15.0 mg/ml 8.0 mg/ml 8.1 mg/ml
Redistilled Freon TF Control without Freon 0.58 g/kg Body wt 2.9 g/kg Body wt
106
MAHURIN AND BERNSTEIN
,5[
<
O X
1.0 i
09 }Z Err W > LU r~
\
/' / / ,//
i
.51
\ /'
\\
2
4
6 8 10
20
/x9 AMINOFLUORINE FIG. 3. Mutagen activation by mouse liver extracts after Freon inhalation: dose-response with aminofluorene. Mice breathing industrial grade Freon TF for 8 hr: 20,000 ppm Freon in air (~); controls, air only (0).
neurochemical changes including decreased cerebral glutathione in rats breathing Freon T E A more recently completed 2-year joint study by DuPont and Allied Corporation has concluded that Freon 113 has no carcinogenic, mutagenic, or teratogenic effect in rats which inhale it at 20,000 ppm for 30 hr per week (as reported in Chemical and Engineering News, July 29, 1985). The work reported here shows that the fluorocarbon Freon TF can enhance the metabolic activation of chemical mutagens. Since the active mutagens are also the proximate or ultimate carcinogenic forms of known chemical carcinogens, this work suggests that low-molecular-weight fluorocarbons may be cocarcinogens. Further work to test the possible cocarcinogenicity of Freons might include these in vitro assays: (1) measure the activity of mixed-function oxidases and cytochrome P-450, in both normal tissue microsomal extracts and tissue extracts from animals exposed to Freon, (2) add Freon directly to tissue extracts, then assay, (3) measure production of polyaromatic hydrocarbon metabolites using HPLC, in tissue extracts with and without Freon. The latter assays might be done with methylcholanthrene, biphenyl, benzo[a]pyrene, or especially 2-aminofluorene or acetylaminofluorene as substrate, which would correlate with the in vivo assays reported here. Extracts of lung tissue from animals breathing Freon TF may be considered most relevant to study, as lung is the organ most exposed to Freons in normal use. However, liver microsomal extracts are often more active and more tractable to achieve, especially considering the requirement for sterility to do mutagenicity tests, as was done here. Further research in these directions is needed to explore the specific biological interactions of Freons with mammalian metabolism.
FLUOROCARBON-ENHANCED MUTAGENESIS
107
CONCLUSIONS Although the degree of enhancement of microsomal activation of chemical carcinogens by brief exposures to low-molecular-weight fluorocarbons may be small, the chronic health risks may be substantial. Studies with cigarette smokers have suggested that several mutational events must occur in humans before neoplastic growth is triggered (reviewed in Cairns, 1981). In smokers and others exposed to ambient low levels of carcinogens, fluorocarbons may significantly increase the risks of cancer in a lifetime by increasing the endogenous level of the active form of the carcinogens. Both smokers and nonsmokers, of course, may be at similar risks from ambient carcinogens other than cigarette smoke. Significant quantities of low-molecular-weight fluorocarbons are produced and used yearly. Our data suggest that fluorocarbons have potential cocarcinogenic activity. We urge a more detailed assessment of the effects of low-molecular-weight fluorocarbons on mammalian metabolism with respect to their cocarcinogenic activity.
REFERENCES Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 347-364. Back, K. C., and van Stee, E. W. (1977). Toxicology of haloalkane propellants and fire extinguishants. Annu. Rev. Pharmacol. Toxicol. 17, 83-95. Bernstein, R. L., Serratto, K. M., and Winant, R. C. (1979). "Chlorinated Insecticides Enhance the Metabolic Activation of Chemical Carcinogens." p. 120. Abstracts of the American Society for Microbiology. Cairns, J. (1981). The origins of human cancers. Nature (London) 289, 353-357. Conaway, C. C., Madhukar, B. V., and Matsumura, E (1977). p,p'-DDT: Studies on induction mechanisms of microsomal enzymes in rat liver systems, Environ. Res. 14, 305-321. Corbett, T. H. (1977). "Cancer and Chemicals." Nelson-Hall, Chicago. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. Magee, E N. (1974). Activation and inactivation of chemical carcinogens and mutagens in the mammal. In "Essays in Biochemistry." (N. Campbell and E Dickens, Eds.), Vol. 10, pp. 105-136. Academic Press, New York/London. Mahurin, R. G. (1980). "Chemical Antimutagenesis and Fluorocarbon Enhancement of Carcinogen Activation." Master's thesis, San Francisco State University. Maugh, T. H. (1979). Blood substitute passes its first test. Science 206, 205. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N. (1975). Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sei. USA 71,979-983. Reinhardt, C. E, Mullin, L. S., and Macfield, M. E. (1972). "Halocarbon-Epinephrine Induced Cardiac Arrhythmia Potential of Some Common Industrial Solvents." 1lth Annual Meeting, Society of Toxicology, Williamsburg, Virginia, March 5-9, 1972. Ryan, D. E., Thomas, E E., Korenionski, D., and Levin, W. (1979). Separation and characterization of highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene. J. Biol. Chem. 254, 1365-1374. Savolainen, H., and Pfaffii, E (1980). Dose-dependent neurochemical effects of 1,1,2-trichloro-l,2,2trifluoroethane inhalation exposure in rats, Toxicol. Lett. 6, 43-49. Turio, R. E (1985). Stratospheric ozone perturbations. In "Ozone in the Free Atmosphere," (R. C. Whitten and S. S. Prasad, Eds.), pp. 195-242. Reinhold, New York. Vainio, H., Nichols, J., and Heinonen, T. (1980). Dose-related hepatotoxicity of 1,1,2-trichloro-l,2,2trifluoroethane in short-term intermittent inhalation exposure in rats. Toxicology 18, 17-25. Waskell, L. (1978). A study of the mutagenicity of anesthetics and their metabolites, Mutat. Res. 57, 141-153.
Fluorocarbon-Enhanced Mutagenesis of Polyaromatic Hyd rocarbons R. G. MAHURIN AND R. L. BERNSTEIN Department of Biological Sciences, San Francisco State University, 1600 Holloway Avenue, San Francisco, California 94132 Received February 24, 1987 The widely used fluorocarbon refrigerant and cleaning solvent 1,1,2-trichloro-l,2,2-trifluoroethane (Freon TF), 1 though generally considered biologically inert, enhances the metabolic activation of chemical carcinogens. Liver microsomal extracts from mice given single intraperitoneal injections of this fluorocarbon showed significant increases in their ability to activate carcinogenic polyaromatic hydrocarbons to form mutagens, compared to control mice injected with saline. Polyaromatic hydrocarbons aminofluorene and acetylaminofluorene were activated in this way. Mutagenicity was measured by a microbial assay. Both commercial grade and redistilled fluorocarbons gave similar results, that is, more highly active liver extracts after administration of the fluorocarbon preparation to mice. Neither industrial grade nor redistilled preparation was itself mutagenic. A combined liver microsomal extract from mice breathing Freon TF at 20,000 ppm in air for 8 hr also had enhanced ability to activate aminofluorene as a mutagen. Exposing mice to Freon TF by inhalation more closely matches the normal route of human exposure to fluorocarbons. The results of this study imply that low-molecular-weight fluorocarbons may pose a carcinogenic risk by acting as cocarcinogenic enhancers of carcinogen activation. The possibility that fluorocarbons are cocarcinogens in this way has apparently not been heretofore considered. © 1988AcademicPress, Inc.
INTRODUCTION Many xenobiotics, often synthetic and toxic chemical substances which are not normally associated with biological organisms, play several roles in initial carcinogenic events. Some of them are frank carcinogens ("initiators"), probably because they are mutagens or are easily activated to become mutagens by mammalian metabolism. Benzo[a]pyrene and acetylaminofluorene are examples of frank xenobiotic carcinogens. Mixed-function oxidases and other enzyme systems are responsible for the metabolism of some xenobiotics which activates them to become carcinogens (Magee, 1974). Some xenobiotics may be cocarcinogens, or substances that are not carcinogenic themselves but act synergistically with frank carcinogens to cause cancer. Two general modes of cocarcinogenesis may be stated. Cocarcinogenesis by "promotion" is one mode, which may involve the stimulation of cell growth after "initiation" or the initial mutagenic lesion has occurred. Presumably croton oil (phorbol esters), saccharin, and some hormones like diethylstilbestrol (DES) are examples of cocarcinogenic promoters. Polychlorinated biphenyls (PCBs), 3-
t Freon TF is a trademark of E. I. DuPont de Nemours & Co. 101 0013-9351/88 $3.00 Copyright© 1988by AcademicPress, Inc. All rightsof reproductionin any formreserved.
102
MAHURIN AND BERNSTEIN
methylcholanthrene, and chlorinated insecticides like DDT and lindane may have another mode of cocarcinogenicity. These latter xenobiotics have been shown to increase the activity of the microsomal enzymes responsible for the generation of other carcinogens by endogenous metabolism (Ryan et al,, 1979, Bernstein et al., 1979). Although low-molecular-weight fluorocarbons have been determined to be relatively harmless by numerous short-term tests over the past 30 years, they have some characteristics that are similar to those of known cocarcinogens. For example, they are synthetic, halogenated, and relatively unreactive chemically. They are also extremely nonpolar. In the liver and other tissues, the cytochrome P-450 system and mixed-function oxidases typically oxidize, hydroxylate, and conjugate many nonpolar compounds to aid in their solubilization and excretion. Many nonpolar compounds (including saturated fats) stimulate the activity of the microsomal oxidizing system (Conaway et al., 1977). Currently in the United States and other industrialized countries large quantities of low-molecular-weight fluorocarbons are produced and used in a wide range of products and processes ranging from refrigerants to washing of silicon chips. Some fluorocarbons ("perfluorochemical emulsions") have been used as blood substitutes (Maugh, 1979). Fluorocarbons are a well-known threat to the atmospheric ozone layer (Turio, 1985). Some anesthetic fluorocarbon gases have been implicated as carcinogens and teratogens (Corbett, 1977), though they are apparently not mutagenic (Waskell, 1978). Another hazard, unsuspected until now, exists if the low-molecular-weight fluorocarbons in widespread use are also significantly cocarcinogenic. One mechanism by which fluorocarbons may be hazardous in this way is their enhancement of enzymatic activation of carcinogenic chemicals. The present paper provides evidence for the fluorocarbon-enhanced activation of polyaromatic hydrocarbons as mutagens. METHODS Bacterial strain Salmonella typhimurium LT-2 TA98 responds well to induced mutation by polyaromatic hydrocarbon mutagens which cause frameshifts (McCann et al., 1975). Tester strain TA100 is isogenic with TA98 except that it is a sensitive indicator of substitution mutations. These bacteria are histidine auxotrophs but revert at a low rate to histidine prototrophy and grow as revertant colonies on minimal medium lacking histidine. In our experiments premutagens aminofluorene and acetylaminofluorene were incorporated into the soft agar overlay during incubation, as described by Ames et al. (1975). These compounds are converted to active mutagens by an extract (called $9) of mammalian liver which is also added to the agar overlay. The bacterial tester strains when exposed to mutagens give increased colony counts as an indicator of active mutagen produced. Microsomal $9 extracts from mammalian liver (here mouse liver) are included in the assay. The microsomal
FLUOROCARBON-ENHANCED MUTAGENESIS
103
enzymatic activities in $9 form highly active mutagenic metabolites of carcinogenic chemicals like acetylaminofluorene and benzo[a]pyrene, which would otherwise not be significantly mutagenic. In the studies reported here mutagenicity of activated forms of aminofluorene and acetylaminofluorene is measured with tester strain TA98. Weanling male Swiss Webster mice (Simonsen Laboratories) received 0.58 or 2.9 g Freon TF1/kg body wt by intraperitoneal injection. Control animals received 0.10 ml of sterile 0.85% NaC1 instead of Freon. Freon TF cleaning solvent (1,1,2trichloro-l,2,2-trifluoroethane) was obtained from DuPont de Nemours & Co. Crude Freon TF is a clear yellowish liquid which boils at 47-49°C. We redistilled Freon TF in a hot water bath with water-cooled condenser, discarding the first and last 10th fractions of distillate. RedistiUed Freon TF is colorless and boils at 48-49 °. Seven days after a single injection of either crude or redistilled Freon TF, livers were removed aseptically. Microsomal $9 extracts were prepared, stored, and used as described (Ames et al., 1975). The same volume, 0.5 ml, of $9 mix containing $9 extract and cofactors was added to each plate in each experiment. The fraction of $9 in $9 mix was held constant at 10%, except as indicated in Fig. 2. Total protein concentrations were determined by the method of Lowry et al. (1951). An additional two groups of five mice each were used to study the effects of inhaling Freon TF. One group breathed crude Freon TF at 20,000 ppm by volume in air for 1 to 2-hr periods, in an enclosed transparent gastight box of volume approx. 5 liters. Total exposure was 8 hr. One group was treated identically but without Freon. Livers were extracted and $9 was prepared as above. The activity of the combined liver extracts of each group of mice toward activating aminofluorene as a mutagen was then tested in a dose-response experiment as described above. RESULTS
No significant difference in body weight or liver weight occurred after 7 days (between controls and Freon-injected animals) as a result of single intraperitoneal injections of Freon TE The fluorocarbon is not itself mutagenic when tested against Salmonella bacterial strains TA98 and TA100 with or without $9 microsomal extract (data not shown here; results and further references are presented in detail in Mahurin, 1980). Freon TF does, however, have an effect on mutagenesis by carcinogenic aromatic hydrocarbons, as shown in Figs. 1 and 2 for tester strain TA98. Liver extracts from mice given a single intraperitoneal injection of industrial grade Freon TF are found to be considerably more active than liver extracts from mice receiving sterile saline, as seen in Fig. 1. Redistilled Freon TF gave similar results in enhancing liver enzymatic activation of carcinogenic chemicals to mutagenic forms (Mahurin, 1980). The protein concentration of $9 liver extracts from mice receiving Freon TF is considerably less than that of controls (Table 1), which means that less $9 protein,
104
MAHURIN AND BERNSTEIN I
J ¢0 O
x },z ,< I.-. cc ILl > IJJ cc
/
1 2 #g
4
/
8
16
2-ACETYLAMINOFLUORINE
FIG. l. Mutagen activation by liver extracts from Freon-injected mice: dose-response with acetylaminofluorene. Intraperitoneal injection of industrial grade Freon TF: 0.58 g/kg body wt (A) or 2.9 g/kg body weight (I). Control mice injected with 0.10 ml sterile 0.85% saline (O). Each data point is the average of three plate counts.
not more, is present in plates containing $9 from Freon-treated mice, yet the extracts are more capable of activating carcinogens. Varying the amount of carcinogen with constant microsomal protein (Fig. I), or varying microsomat protein with a constant amount of carcinogen (Fig. 2), had similar effects. In all cases liver extracts from mice injected with Freon TF were more active than extracts from mice injected with sterile saline. The number of revertants using Freon-activated mouse liver $9 reached values similar to those we obtained using Aroclor 1254-activated rat liver, namely 2000-3000 revertants per plate at doses of 8-12 ~g acetylaminofluorene or 600-800 p~g aminofluorene per plate (Mahurin, 1980). Liver extracts from mice exposed to 20,000 ppm Freon TF by volume in air had increased ability to generate active mutagens when compared to mice breathing air only (Fig. 3). We found no significant change in total body weight or weight of lungs or livers from Freon exposure by inhalation. The data of Fig. 3 indicate that the activation of small amounts of aminofluorene for bacterial mutagenesis is especially greatly enhanced by $9 extract of mice breathing Freon TE DISCUSSION
The American Conference of Governmental Industrial Hygienists has assigned a safety threshold limit for inhalation of 1000 ppm by volume for Freon TF in air for a time-weighted average workday. This is the highest limit allowable for any chemical under the current guidelines, and it implies that Freons are relatively
FLUOROCARBON-ENHANCED MUTAGENESIS
41
A
7 jj~.
105
B
6
O9 O
5
X (D t'Z < t,¢r" LLI > LLI or
CO O 4
X I.,Z< I--
3
W
>
2
W rr'
i
0.50
1.00
1.50
0J50
1.00
1.~50
MG PROTEIN PER PLATE MG PROTEIN PER PLATE
FIG. 2. Mutagen activation by varying amounts of mouse liver $9 extract. Intraperitoneal injection of industrial grade (A) or redistilled (B) Freon TF: symbols as in Fig. 1. All plates contained 600 ~g 2-aminofluorene. Each data point is the average of three plate counts.
safe. However, Vainio et al. (1980) found Freon TF to be a hepatotoxin in rats. Changes in smooth endoplasmic reticulum accompanied alterations in microsomal enzyme activities. They also found cytochrome P-450 binds Freon TE In our study mice given intraperitoneal injections of Freon TF also showed characteristically altered livers. Exposure to haloalkanes is known to cause cardiac malfunctions in animals at high doses (Back and van Stee, 1977). Reinhardt et al. (1972) discovered that trichlorotrifluoroethane (Freon 113) causes cardiac sensitization in mammals at concentrations of 5000 ppm by volume in air. Savolainen and Pf~iffli (1980) noted TABLE l PROTEIN CONCENTRATIONS IN LIVER $9 MICROSOMAL EXTRACTS
Mice injected with
No. of animals
Average protein in extract
Commercial grade Freon TF Control without Freon 0.58 g/kg Body wt 2.9 g/kg Body wt
12 12 8
14.0 mg/ml 8.6 mg/ml 7.5 mg/ml
5 6 6
15.0 mg/ml 8.0 mg/ml 8.1 mg/ml
Redistilled Freon TF Control without Freon 0.58 g/kg Body wt 2.9 g/kg Body wt
106
MAHURIN AND BERNSTEIN
,5[
<
O X
1.0 i
09 }Z Err W > LU r~
\
/' / / ,//
i
.51
\ /'
\\
2
4
6 8 10
20
/x9 AMINOFLUORINE FIG. 3. Mutagen activation by mouse liver extracts after Freon inhalation: dose-response with aminofluorene. Mice breathing industrial grade Freon TF for 8 hr: 20,000 ppm Freon in air (~); controls, air only (0).
neurochemical changes including decreased cerebral glutathione in rats breathing Freon T E A more recently completed 2-year joint study by DuPont and Allied Corporation has concluded that Freon 113 has no carcinogenic, mutagenic, or teratogenic effect in rats which inhale it at 20,000 ppm for 30 hr per week (as reported in Chemical and Engineering News, July 29, 1985). The work reported here shows that the fluorocarbon Freon TF can enhance the metabolic activation of chemical mutagens. Since the active mutagens are also the proximate or ultimate carcinogenic forms of known chemical carcinogens, this work suggests that low-molecular-weight fluorocarbons may be cocarcinogens. Further work to test the possible cocarcinogenicity of Freons might include these in vitro assays: (1) measure the activity of mixed-function oxidases and cytochrome P-450, in both normal tissue microsomal extracts and tissue extracts from animals exposed to Freon, (2) add Freon directly to tissue extracts, then assay, (3) measure production of polyaromatic hydrocarbon metabolites using HPLC, in tissue extracts with and without Freon. The latter assays might be done with methylcholanthrene, biphenyl, benzo[a]pyrene, or especially 2-aminofluorene or acetylaminofluorene as substrate, which would correlate with the in vivo assays reported here. Extracts of lung tissue from animals breathing Freon TF may be considered most relevant to study, as lung is the organ most exposed to Freons in normal use. However, liver microsomal extracts are often more active and more tractable to achieve, especially considering the requirement for sterility to do mutagenicity tests, as was done here. Further research in these directions is needed to explore the specific biological interactions of Freons with mammalian metabolism.
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CONCLUSIONS Although the degree of enhancement of microsomal activation of chemical carcinogens by brief exposures to low-molecular-weight fluorocarbons may be small, the chronic health risks may be substantial. Studies with cigarette smokers have suggested that several mutational events must occur in humans before neoplastic growth is triggered (reviewed in Cairns, 1981). In smokers and others exposed to ambient low levels of carcinogens, fluorocarbons may significantly increase the risks of cancer in a lifetime by increasing the endogenous level of the active form of the carcinogens. Both smokers and nonsmokers, of course, may be at similar risks from ambient carcinogens other than cigarette smoke. Significant quantities of low-molecular-weight fluorocarbons are produced and used yearly. Our data suggest that fluorocarbons have potential cocarcinogenic activity. We urge a more detailed assessment of the effects of low-molecular-weight fluorocarbons on mammalian metabolism with respect to their cocarcinogenic activity.
REFERENCES Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 347-364. Back, K. C., and van Stee, E. W. (1977). Toxicology of haloalkane propellants and fire extinguishants. Annu. Rev. Pharmacol. Toxicol. 17, 83-95. Bernstein, R. L., Serratto, K. M., and Winant, R. C. (1979). "Chlorinated Insecticides Enhance the Metabolic Activation of Chemical Carcinogens." p. 120. Abstracts of the American Society for Microbiology. Cairns, J. (1981). The origins of human cancers. Nature (London) 289, 353-357. Conaway, C. C., Madhukar, B. V., and Matsumura, E (1977). p,p'-DDT: Studies on induction mechanisms of microsomal enzymes in rat liver systems, Environ. Res. 14, 305-321. Corbett, T. H. (1977). "Cancer and Chemicals." Nelson-Hall, Chicago. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. Magee, E N. (1974). Activation and inactivation of chemical carcinogens and mutagens in the mammal. In "Essays in Biochemistry." (N. Campbell and E Dickens, Eds.), Vol. 10, pp. 105-136. Academic Press, New York/London. Mahurin, R. G. (1980). "Chemical Antimutagenesis and Fluorocarbon Enhancement of Carcinogen Activation." Master's thesis, San Francisco State University. Maugh, T. H. (1979). Blood substitute passes its first test. Science 206, 205. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N. (1975). Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sei. USA 71,979-983. Reinhardt, C. E, Mullin, L. S., and Macfield, M. E. (1972). "Halocarbon-Epinephrine Induced Cardiac Arrhythmia Potential of Some Common Industrial Solvents." 1lth Annual Meeting, Society of Toxicology, Williamsburg, Virginia, March 5-9, 1972. Ryan, D. E., Thomas, E E., Korenionski, D., and Levin, W. (1979). Separation and characterization of highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene. J. Biol. Chem. 254, 1365-1374. Savolainen, H., and Pfaffii, E (1980). Dose-dependent neurochemical effects of 1,1,2-trichloro-l,2,2trifluoroethane inhalation exposure in rats, Toxicol. Lett. 6, 43-49. Turio, R. E (1985). Stratospheric ozone perturbations. In "Ozone in the Free Atmosphere," (R. C. Whitten and S. S. Prasad, Eds.), pp. 195-242. Reinhold, New York. Vainio, H., Nichols, J., and Heinonen, T. (1980). Dose-related hepatotoxicity of 1,1,2-trichloro-l,2,2trifluoroethane in short-term intermittent inhalation exposure in rats. Toxicology 18, 17-25. Waskell, L. (1978). A study of the mutagenicity of anesthetics and their metabolites, Mutat. Res. 57, 141-153.