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Comparison of lung injury induced in 4 strains of mice by butylated hydroxytoluene
Toxicology Letters. 52 (1990) 55561 Elsevier
TOXLET
02338
Comparison of lung injury induced in 4 strains of mice by butylated hydroxytoluene
James P. Kehrer’ and John DiGiovanni2 ‘Division of Pharmacology
and Toxicology,
College of Pharmacy,
Austin, TX and ZDepartment of Carcinogenesis,
The University of Texas at Austin,
The University of Texas M.D. Anderson Cancer Center,
Science Park, SmithviNe, TX (U.S.A.) (Received
22 September
(Accepted
9 February
Ke.v words; Butylated
1989) 1990)
hydroxytoluene;
Lung damage;
Pulmonary
fibrosis; Mouse strains
SUMMARY Butylated mace which
hydroxytoluene
(BHT) is a phenolic
have been tested,
also highly strain-dependent, doses. the relationship demonstratethat exhrbit
similar
with LDS,s ranging
between
BALB/c,
lung damage
of repair
from 138 to 1739 mg/kg.
(as assessed
by the incorporation
fibrosis (gs assessed by lung hydroxyproline
of repair when given a single dose of 300 mg/kg that all strains
that the extent of lung damage
in all strains
of
with BHT is
Despite this wide range of toxic The data presented
similar
of radiolabelled
content)
350 mg/kg,
BHT, although
of mice develop
produced
lung injury
of mice treated
here
mice, with LD%s of 1739, 1243 and 917 respectively,
dose of BHT. SSIn mice, with an LDwl of approximately These data indicate
which induces
species. The mortality
and dose has not been well studied.
ICR and C57BL/6NHsd
time courses
DNA) and pulmonary
antioxidant
but not in any other
also exhibited
into
a similar time course
fibrosis did not develop
levels of lung injury
in mice does not correlate
thymidine
when given a single 400 mg/kg in these animals.
at equivalent
doses and
with the lethal dose.
INTRODUCTION
Butylated hydroxytoluene (BHT) has been found to elicit a diffuse lung lesion in both male and female mice but not other species including rat, hamster and rabbit [l-3; unpublished data]. The reason for this species selectivity and the mechanism of the lung injury are not certain, but recent data strongly implicate differences in metabolic pathways, both activation and deactivation. The metabolic activation of Address for correspondence: The University
James
of Texas at Austin,
P. Kehrer, Austin,
Ph.D.,
Division
TX 78712-1074,
of Pharmacology,
U.S.A.
Phone:
471-8762.
0378-4274/90/S
3.50 @ 1990 Elsevier Science Publishers
B.V. (Biomedical
College
(512) 471-1107;
Division)
of Pharmacy, FAX: (512)
56
BHT has been shown to be responsible for lung injury in mice [3] and it appears likely that a BHT-quinone methide is the specific metabolite responsible for lung injury [4,
51. Although BHT is highly species-specific in its ability to induce lung injury, strain differences in lung injury have not been studied in detail. Witschi et al. [6] reported similar increases in pulmonary DNA synthesis 2 and 4 days after 400 mg/kg BHT in 6 mouse strains and a C57 x C3H Fl hybrid, while Malkinson [7] reported some variability in the extent to which the lung/body weight ratio was increased in 14 strains given 400 mg/kg BHT. Differences between strains in the lung/body weight ratio are suggestive of variations in responsiveness to BHT. However, only a few animals were tested for some strains and the time course and magnitude of the injury and repair process have not been quantitated. There are significant strain differences in mortality following treatment of mice with BHT [S], but it is not clear whether lung damage is involved in the observed deaths. LDsss in mice range from 138 to 1739 mg/kg [8]. This lo-fold range in acute toxicity between strains is highly unusual for a chemical. The purpose of the present study was to assess the time course of lung repair and the extent of pulmonary fibrosis in several mouse strains with a wide range of LD5s.s given the same dose of BHT. The results demonstrate that similar doses of BHT elicit comparable lung injury in all strains, and thus lung injury does not correlate with mortality. MATERIALS
AND METHODS
Chemicals
BHT and DNA (from calf thymus) were obtained from Sigma Chemical Company (St. Louis, MO). [2-‘4C]Thymidine, 52.2 Ci/mol, was obtained from ICN (Irvine, CA). All other chemicals used were reagent or spectrophotometric grade. Animals and treatments
Male BALB/c mice (23-28 g, 8-9 weeks of age), were bred and maintained in the Animal Resources Center at the University of Texas at Austin, Female SSIn mice (2628 g, 9 weeks of age) were bred at the University of Texas M.D. Anderson Cancer Center in Smithville, TX [9]. Male ICR (25-30 g, 9 weeks of age) and male C57BL/6NHsd mice (2&25 g, 9 weeks of age) were obtained from Harlan Sprague Dawley (Houston, TX). BHT was dissolved in corn oil before being administered to mice intraperitoneally. Drug concentrations were adjusted so that mice received 0.1 ml/10 g body wt. Control mice received an equal volume of corn oil. Food and water were available ad libitum. Mice were euthanized by cervical dislocation. Pulmonary
DNA synthesis
DNA synthesis was assessed as an index of cell proliferation (and indirectly as a measure of the extent of the damage) [3] by measuring the incorporation of thymidine
into total lung DNA. Mice were given an intraperitoneal injection of 0.5 ,&i [14C]thymidine, euthanized after 90 min and the specific activity of pulmonary DNA was determined [lo]. Briefly, each individual lung was removed and homogenized in 3 ml water. The homogenate was immediately mixed with 2 ml 0.5 N HC104 and centrifuged. The resultant pellet was washed twice with 5 ml cold 0.5 N HC104, then digested at 70°C for 20 min in 4 ml 1.5 N HC104. This mixture was cooled, centrifuged and 2 ml of the resultant supernatant counted for radioactivity by liquid scintillation. A 0.2 ml aliquot of each supernatant fraction was assayed for total DNA using Richards’ modification of the diphenylamine reaction [ 1I]. Hydroxyproline analysis
Total lung collagen was estimated by measuring hydroxyproline, an amino acid found primarily in collagen [12], 21 days after treatment. Lung tissue was excised, lyophilized and hydrolyzed for 18 h at 107°C in 4 ml of 6 N HCl. The hydrolysate was then neutralized with 10 N NaOH and assayed for hydroxyproline by the method of Witschi et al. [ 131. Statistics
All data are expressed as means + SE. The standard deviations of the thymidine incorporation data were found to be directly proportional to the means of each group. These data were therefore transformed by taking their natural logarithm before analysis by a one-way analysis of variance [14]. All multiple group data were analyzed by one-way ANOVA program with Student-Neuman-Keuls post-hoc comparisons [14]. A P-value of < 0.05 was considered significant for all experiments. RESULTS
LD5,, values previously reported for BALB/c AnN, ICR-JCL and C57BL/6N mice were 1739, 1243 and 917 mg/kg, respectively [8]. We did not repeat these findings in our particular animals, but noted no mortality at 400 mg/kg in BALB/c mice, and only 10 and 14% in the ICR and C57 strains, respectively. LDsos have not been reported in SSIn mice and were also not determined in the current study. However, 200, 300 and 400 mg/kg resulted in 0, 8 and 100% mortality, respectively, suggesting that the LDSs dose is approximately 350 mg/kg. Some minor strain differences were evident in control pulmonary DNA synthesis. BALB/c, ICR and SSIn mice were not different, incorporating 543 +41 (n = 9), 462 + 28 (n = 9) and 339 f 55 (n = 6) dpm/mg DNA, respectively. However, C57 mice at 974 + 133 (n = 7) dpm/mg DNA exhibited a somewhat higher basal level of pulmonary DNA synthesis. Differences between strains were also evident in thymidine incorporation at various days after a single 400 mg/kg dose of BHT (Fig. 1). BALB/c mice increased their rate of pulmonary DNA synthesis more rapidly than the other strains, but reached lower peak levels. C57 mice exhibited the highest level of thymi-
58 20000
0 control Fig.
1.Time course of pulmonary
300 or 400 mg/kg
2 thymidine
BHT. Data are expressed
3
4 I Time (days)
incorporation
after treatment
at these different
days,
14
of different
as mean & SE. n =4 or 5 for all treatment
were run on days 2, 7 and 14 (2 and 7 for SSIn mice) after iqection differences
10
so the data
were combined.
strains of mice with days. Three controls
of corn oil. There were no significant *Significantly
different
from
controls
(PCO.05).
dine incorporation, suggesting that this strain developed the most lung injury. C57 mice also had the most prolonged repair process, reaching a peak at day 7 and not returning to control levels until 14 days after the initial treatment. Increases in pulmonary thymidine incorporation in ICR mice reached maximal levels 4 days after treatment and then rapidly declined to control levels by day 10, indicative of a somewhat more rapid repair process. However, data from this strain also had the greatest variability, perhaps because of their outbred nature. SSIn mice treated with 300 mg/kg BHT exhibited increases in pulmonary DNA synthesis nearly as large as in C57 and ICR mice given 400 mg/kg BHT, and exceeded that of BALB/c mice given this higher dose. Since the higher dose resulted in death of all SSIn mice, more direct comparisons could not be made. Increases in total lung hydroxyproline content 21 days after treatment with BHT were evident in BALB/c, ICR and C57 mice (Fig. 2). The magnitude of these increases was the same in all strains despite differences in the basal content of lung BALB/c = 243 + 14 pug/lung (n = 6); ICR = 391+ 19 (n = 5); hydroxyproline. C57 = 287 f 8 (n = 5). In contrast, SSIn mice (basal levels 335 + 12 (n = 9)) did not have an elevated lung content of hydroxyproline despite evidence from the thymidine incorporation studies of comparable lung injury. DISCUSSION
BHT-induced lung injury in mice has been extensively studied at the pathological level. Although the cause of death in mice given high doses of this chemical has not
59
Mouse Strains Fig. 2. Total
lung hydroxyproline
21 days after treatment
are expressed
as the mean percentage
BALB/c=243+
14 (6): ICR=391+
of control
f
19 (5); (X7=287*8
ses = n. *Significantly
different
of different
SE. Control
strains
of mice with BHT.
values were (in mean &lung
(5); SSIn=335+ from controls
12 (9). Values
f
Data SE):
in parenthe-
(P < 0.05).
been established, it has been assumed to be related to the lung damage [15]. BHT damages mouse lung tissue regardless of strain, and this injury appears to be of a similar magnitude at equivalent doses [6]. This finding is surprising since different mouse strains exhibit dramatically different LDSOS [8]. There is a dose-dependent relationship between BHT and pulmonary DNA synthesis at low doses, but this appears to reach a maximal level long before mortality is observed, at least in BALB/c mice [16]. A similar maximization of response appears in DBA and C57 mice when lung weight is used as the index of injury [8]. Thus, it seems unlikely that lung damage is the proximal cause of death unless the indices used to quantitate injury reach maximal responses before maximal injury is achieved and therefore fail to be linearly related to dose. In the current study, the time course and extent of lung injury and repair was examined in 4 mouse strains with widely different LDses (350-1700 mg/kg). The incorporation of radiolabelled thymidine into pulmonary DNA, which has been related to the extent of the initial injury [3, 171 was used as an index of damage. Control levels of pulmonary DNA synthesis were similar in all strains except C57 mice. The slightly higher levels seen in this strain may have been related to their smaller body size. All mice received 0.5 @i thymidine, and in smaller animals this should result in a higher specific activity. Despite the wide range of doses needed to produce death, the time course of lung cell division after BHT was generally the same between strains given identical doses. This included doses of from 23% (BALB/c) tox85% (SSIn) of the LDs,-, values. In addition, a previous report in Swiss-Webster mice given 400 mg/kg BHT demonstrated a similar time course of injury and repair [7]. Some subtle differences may exist between strains, as demonstrated by the slightly more rapid repair in BALB/c and
60
ICR mice, but these differences seem unlikely to have any physiological significance. The development of pulmonary fibrosis, as reflected by lung hydroxyproline content, also failed to reveal any strain-related differences in responsiveness to BHT-induced damage. BALB/c, ICR and C57 mice had similar percentage increases in lung fibrosis which were far from the maximum achievable in these animals. Differences in absolute increases were observed, but seemed primarily due to differences in body weight and thus lung size before treatment. BHT-induced increases in pulmonary hydroxyproline were not detected in SSIn mice. This may have been related to the lower absolute dose given to these mice (300 vs. 400 mg/kg BHT), although the dose relative to the LDs,-,was the highest in this strain. Previous work has indicated a threshold dose for the development of fibrosis in mice treated with BHT [lo], and it is possible the dose given to SSIn mice did not exceed this threshold despite the obvious injury produced. Interestingly, there appears to be a maximal level of fibrosis produced by BHT in mice. Differences between mouse strains in their sensitivity to skin tumor promotion using phorbol esters have recently been related to the ability of skin tissue to undergo a sustained hyperplasiagenic response [18]. Among the mouse strains used in the present study, SSIn and DBA/2 are highly sensitive, ICR are intermediate, and BALB/c and C57BL/6 are relatively resistant to phorbol ester promotion [18]. In general, strains which exhibit higher levels of sustained hyperplasia are more susceptible to tumor promotion by phorbol esters [18]. Furthermore, a recent study demonstrated that SENCAR mice (the progenitor of SSIn) exhibited an exaggerated and persistent epidermal hyperplasia in response to tissue damage caused by ultraviolet irradiation compared to BALB/c mice [19]. One rationale for the current study was to determine if a similar relationship might hold for chemical-induced cell proliferation of internal organs. The results demonstrate, however, that the time course of repair after BHT-induced lung damage was basically similar in all strains and the minor differences which were observed do not correlate with the susceptibility of these strains to skin tumor promotion or ultraviolet-induced carcinogenesis. Thus, strains of mice with different susceptibilities to phorbol ester promotion, including the selectively bred SSIn, do not appear to differ significantly in their response to agents toxic for the lung, such as BHT. BHT has been found to both inhibit and promote tumor formation in different model systems [2]. Strain differences in the effect of BHT on urethane-induced mouse lung adenomas have been reported [20]. The mechanism of these differences has not been explored. Correlations between these differences and BHT metabolism, lung injury and overall mortality would be of interest and may provide insights into the mechanism by which BHT can function as a tumor promoter. In summary, BHT-induced lung injury is evident in all strains of mice which have been tested. The magnitude of this injury shows only minor variations between strains given equivalent doses. Thus, the overall toxicity of BHT to the different strains of mice appears not to be related to the extent of lung damage induced by this antioxidant.
61 ACKNOWLEDGEMENTS
This work was supported by NIH grants HL35689 (J.P.K.) and CA38871 (J.D.). J.P.K. is the recipient of Research Career Development Award HL01435. Thanks go to Mr. Thomas Devor for his excellent technical assistance. REFERENCES
1 Miyakawa, Y., Takahashi, M., Furukawa, F., Toyoda, K., Sato, H. and Hayashi, Y. (1986) Pneumotoxicity of butylated hydroxytoluene applied dermally to CD-l mice. Toxicol. Lett. 34,9%105. 2 Witschi, H., Malkinson, A.M. and Thompson, J.A. (1989) Metabolism and pulmonary toxicity of butylated hydroxytoluene (BHT). Pharmacol. Ther., 42,89-l 13. 3 Kehrer, J.P. and Witschi, H. (1980) Effects of drug metabolism inhibitors on butylated hydroxytoluene-induced pulmonary toxicity in mice. Toxicol. Appl. Pharmacol. 53,333-342. 4 Mizutani, T., Yamamoto, K. and Tajima, K. (1983) Isotope effects on the metabolism and pulmonary toxicity of butylated hydroxytohtene in mice by deuteration of the 4-methyl group. Toxicol. Appl. Pharmacol. 69,283-290. 5 Mizutani, T., Nomura, H., Yamamoto, K. and Tajima, K. (1984) Modification of butylated hydroxytoluene-induced pulmonary toxicity in mice by diethyl maleate, buthionine sulfoxime, and cysteine. Toxicol. Lett. 23, 327-331. 6 Witschi, H.P., Kacew, S., Tsang, B.J. and Williamson, D. (1976) Biochemical parameters of BHT-induced cell growth in mouse lung. Chem.-Biol. Interact., 12.2940. 7 Malkinson, A.M. (1979) Prevention of butylated hydroxytoluene-induced lung damage in mice by cedar terpene administration, Toxicol. Appl. Pharmacol. 49, 551-560. 8 Kawano, S., Nakao, T. and Hiraga, K. (1981) Strain differences in butylated hydroxytoluene-induced deaths in male mice. Toxicol. Appl. Pharmacol. 61,475479. 9 Fischer, S.M., O’Connell, J.F., Conti, C.J., Tacker, K.C., Fries, J.W., Patrick, K.E., Adams, L.M. and Slaga, T.J. (1987) Characterization of an inbred strain of the SENCAR mouse that is highly sensitive to phorbol esters. Carcinogenesis, 8,421424. 10 Kehrer, J.P. (1982) Collagen production rates following acute lung damage induced by butylated hydroxytoluene, Biochem. Pharmacol. 31,2053-2058. 11 Richards, G.M. (1974) Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57,369-376. 12 Hance, A.J. and Crystal, R.G. (1976) Collagen. In: R.G. Crystal (Ed.), The Biochemical Basis of Pulmonary Function. Marcel Dekker, New York, pp. 2 15-27 1. 13 Witschi, H.P., Tryka, A.F. and Lindenschmidt, R.C. (1985) The many faces of an increase in lung collagen. Fundam. Appl. Toxicol. 5,240-250. 14 Zivin, J.A. and Bartko, J.J. (1976) Statistics for disinterested scientists. Life Sci. 18, 15-26. 15 Marino, A.A. and Mitchell, J.T. (1972) Lung damage in mice following intraperitoneal injection of butylated hydroxytoluene, Proc. Sot. Exp. Biol. Med. 140, 122-125. 16 Witschi, H.P. and Lock, S. (1978) Toxicity of butylated hydroxytoluene in mouse following oral administration. Toxicology, 9, 137-146. 17 Evans, M.J., Dekker, N.P., Cabral-Anderson, L.J. and Freeman, G. (1978) Quantitation of damage of the alveolar epithelium by means of type 2 cell proliferation. Am. Rev. Respir. Dis. 118, 787-790. 18 DiGiovanni, J. (1989) Genetic determinants of susceptibility to mouse skin tumor promotion in inbred mice. In N.H. Colbum (Ed.), Genes and Signal Transduction in Multistage Carcinogenesis. Marcel Dekker, New York, pp. 3967. 19 Strickland, P.T. (1986) Abnormal wound healing in uv-irradiated skin of SENCAR mice. J. Invest. Dermatol. 86, 3741. 20 Malkinson, A.M. and Thaete, L.G. (1986) Effects of strain and age on prophylaxis and co-carcinogenesis of urethan-induced mouse lung adenomas by butylated hydroxytoluene. Cancer Res. 46, 16941697.
TOXLET
02338
Comparison of lung injury induced in 4 strains of mice by butylated hydroxytoluene
James P. Kehrer’ and John DiGiovanni2 ‘Division of Pharmacology
and Toxicology,
College of Pharmacy,
Austin, TX and ZDepartment of Carcinogenesis,
The University of Texas at Austin,
The University of Texas M.D. Anderson Cancer Center,
Science Park, SmithviNe, TX (U.S.A.) (Received
22 September
(Accepted
9 February
Ke.v words; Butylated
1989) 1990)
hydroxytoluene;
Lung damage;
Pulmonary
fibrosis; Mouse strains
SUMMARY Butylated mace which
hydroxytoluene
(BHT) is a phenolic
have been tested,
also highly strain-dependent, doses. the relationship demonstratethat exhrbit
similar
with LDS,s ranging
between
BALB/c,
lung damage
of repair
from 138 to 1739 mg/kg.
(as assessed
by the incorporation
fibrosis (gs assessed by lung hydroxyproline
of repair when given a single dose of 300 mg/kg that all strains
that the extent of lung damage
in all strains
of
with BHT is
Despite this wide range of toxic The data presented
similar
of radiolabelled
content)
350 mg/kg,
BHT, although
of mice develop
produced
lung injury
of mice treated
here
mice, with LD%s of 1739, 1243 and 917 respectively,
dose of BHT. SSIn mice, with an LDwl of approximately These data indicate
which induces
species. The mortality
and dose has not been well studied.
ICR and C57BL/6NHsd
time courses
DNA) and pulmonary
antioxidant
but not in any other
also exhibited
into
a similar time course
fibrosis did not develop
levels of lung injury
in mice does not correlate
thymidine
when given a single 400 mg/kg in these animals.
at equivalent
doses and
with the lethal dose.
INTRODUCTION
Butylated hydroxytoluene (BHT) has been found to elicit a diffuse lung lesion in both male and female mice but not other species including rat, hamster and rabbit [l-3; unpublished data]. The reason for this species selectivity and the mechanism of the lung injury are not certain, but recent data strongly implicate differences in metabolic pathways, both activation and deactivation. The metabolic activation of Address for correspondence: The University
James
of Texas at Austin,
P. Kehrer, Austin,
Ph.D.,
Division
TX 78712-1074,
of Pharmacology,
U.S.A.
Phone:
471-8762.
0378-4274/90/S
3.50 @ 1990 Elsevier Science Publishers
B.V. (Biomedical
College
(512) 471-1107;
Division)
of Pharmacy, FAX: (512)
56
BHT has been shown to be responsible for lung injury in mice [3] and it appears likely that a BHT-quinone methide is the specific metabolite responsible for lung injury [4,
51. Although BHT is highly species-specific in its ability to induce lung injury, strain differences in lung injury have not been studied in detail. Witschi et al. [6] reported similar increases in pulmonary DNA synthesis 2 and 4 days after 400 mg/kg BHT in 6 mouse strains and a C57 x C3H Fl hybrid, while Malkinson [7] reported some variability in the extent to which the lung/body weight ratio was increased in 14 strains given 400 mg/kg BHT. Differences between strains in the lung/body weight ratio are suggestive of variations in responsiveness to BHT. However, only a few animals were tested for some strains and the time course and magnitude of the injury and repair process have not been quantitated. There are significant strain differences in mortality following treatment of mice with BHT [S], but it is not clear whether lung damage is involved in the observed deaths. LDsss in mice range from 138 to 1739 mg/kg [8]. This lo-fold range in acute toxicity between strains is highly unusual for a chemical. The purpose of the present study was to assess the time course of lung repair and the extent of pulmonary fibrosis in several mouse strains with a wide range of LD5s.s given the same dose of BHT. The results demonstrate that similar doses of BHT elicit comparable lung injury in all strains, and thus lung injury does not correlate with mortality. MATERIALS
AND METHODS
Chemicals
BHT and DNA (from calf thymus) were obtained from Sigma Chemical Company (St. Louis, MO). [2-‘4C]Thymidine, 52.2 Ci/mol, was obtained from ICN (Irvine, CA). All other chemicals used were reagent or spectrophotometric grade. Animals and treatments
Male BALB/c mice (23-28 g, 8-9 weeks of age), were bred and maintained in the Animal Resources Center at the University of Texas at Austin, Female SSIn mice (2628 g, 9 weeks of age) were bred at the University of Texas M.D. Anderson Cancer Center in Smithville, TX [9]. Male ICR (25-30 g, 9 weeks of age) and male C57BL/6NHsd mice (2&25 g, 9 weeks of age) were obtained from Harlan Sprague Dawley (Houston, TX). BHT was dissolved in corn oil before being administered to mice intraperitoneally. Drug concentrations were adjusted so that mice received 0.1 ml/10 g body wt. Control mice received an equal volume of corn oil. Food and water were available ad libitum. Mice were euthanized by cervical dislocation. Pulmonary
DNA synthesis
DNA synthesis was assessed as an index of cell proliferation (and indirectly as a measure of the extent of the damage) [3] by measuring the incorporation of thymidine
into total lung DNA. Mice were given an intraperitoneal injection of 0.5 ,&i [14C]thymidine, euthanized after 90 min and the specific activity of pulmonary DNA was determined [lo]. Briefly, each individual lung was removed and homogenized in 3 ml water. The homogenate was immediately mixed with 2 ml 0.5 N HC104 and centrifuged. The resultant pellet was washed twice with 5 ml cold 0.5 N HC104, then digested at 70°C for 20 min in 4 ml 1.5 N HC104. This mixture was cooled, centrifuged and 2 ml of the resultant supernatant counted for radioactivity by liquid scintillation. A 0.2 ml aliquot of each supernatant fraction was assayed for total DNA using Richards’ modification of the diphenylamine reaction [ 1I]. Hydroxyproline analysis
Total lung collagen was estimated by measuring hydroxyproline, an amino acid found primarily in collagen [12], 21 days after treatment. Lung tissue was excised, lyophilized and hydrolyzed for 18 h at 107°C in 4 ml of 6 N HCl. The hydrolysate was then neutralized with 10 N NaOH and assayed for hydroxyproline by the method of Witschi et al. [ 131. Statistics
All data are expressed as means + SE. The standard deviations of the thymidine incorporation data were found to be directly proportional to the means of each group. These data were therefore transformed by taking their natural logarithm before analysis by a one-way analysis of variance [14]. All multiple group data were analyzed by one-way ANOVA program with Student-Neuman-Keuls post-hoc comparisons [14]. A P-value of < 0.05 was considered significant for all experiments. RESULTS
LD5,, values previously reported for BALB/c AnN, ICR-JCL and C57BL/6N mice were 1739, 1243 and 917 mg/kg, respectively [8]. We did not repeat these findings in our particular animals, but noted no mortality at 400 mg/kg in BALB/c mice, and only 10 and 14% in the ICR and C57 strains, respectively. LDsos have not been reported in SSIn mice and were also not determined in the current study. However, 200, 300 and 400 mg/kg resulted in 0, 8 and 100% mortality, respectively, suggesting that the LDSs dose is approximately 350 mg/kg. Some minor strain differences were evident in control pulmonary DNA synthesis. BALB/c, ICR and SSIn mice were not different, incorporating 543 +41 (n = 9), 462 + 28 (n = 9) and 339 f 55 (n = 6) dpm/mg DNA, respectively. However, C57 mice at 974 + 133 (n = 7) dpm/mg DNA exhibited a somewhat higher basal level of pulmonary DNA synthesis. Differences between strains were also evident in thymidine incorporation at various days after a single 400 mg/kg dose of BHT (Fig. 1). BALB/c mice increased their rate of pulmonary DNA synthesis more rapidly than the other strains, but reached lower peak levels. C57 mice exhibited the highest level of thymi-
58 20000
0 control Fig.
1.Time course of pulmonary
300 or 400 mg/kg
2 thymidine
BHT. Data are expressed
3
4 I Time (days)
incorporation
after treatment
at these different
days,
14
of different
as mean & SE. n =4 or 5 for all treatment
were run on days 2, 7 and 14 (2 and 7 for SSIn mice) after iqection differences
10
so the data
were combined.
strains of mice with days. Three controls
of corn oil. There were no significant *Significantly
different
from
controls
(PCO.05).
dine incorporation, suggesting that this strain developed the most lung injury. C57 mice also had the most prolonged repair process, reaching a peak at day 7 and not returning to control levels until 14 days after the initial treatment. Increases in pulmonary thymidine incorporation in ICR mice reached maximal levels 4 days after treatment and then rapidly declined to control levels by day 10, indicative of a somewhat more rapid repair process. However, data from this strain also had the greatest variability, perhaps because of their outbred nature. SSIn mice treated with 300 mg/kg BHT exhibited increases in pulmonary DNA synthesis nearly as large as in C57 and ICR mice given 400 mg/kg BHT, and exceeded that of BALB/c mice given this higher dose. Since the higher dose resulted in death of all SSIn mice, more direct comparisons could not be made. Increases in total lung hydroxyproline content 21 days after treatment with BHT were evident in BALB/c, ICR and C57 mice (Fig. 2). The magnitude of these increases was the same in all strains despite differences in the basal content of lung BALB/c = 243 + 14 pug/lung (n = 6); ICR = 391+ 19 (n = 5); hydroxyproline. C57 = 287 f 8 (n = 5). In contrast, SSIn mice (basal levels 335 + 12 (n = 9)) did not have an elevated lung content of hydroxyproline despite evidence from the thymidine incorporation studies of comparable lung injury. DISCUSSION
BHT-induced lung injury in mice has been extensively studied at the pathological level. Although the cause of death in mice given high doses of this chemical has not
59
Mouse Strains Fig. 2. Total
lung hydroxyproline
21 days after treatment
are expressed
as the mean percentage
BALB/c=243+
14 (6): ICR=391+
of control
f
19 (5); (X7=287*8
ses = n. *Significantly
different
of different
SE. Control
strains
of mice with BHT.
values were (in mean &lung
(5); SSIn=335+ from controls
12 (9). Values
f
Data SE):
in parenthe-
(P < 0.05).
been established, it has been assumed to be related to the lung damage [15]. BHT damages mouse lung tissue regardless of strain, and this injury appears to be of a similar magnitude at equivalent doses [6]. This finding is surprising since different mouse strains exhibit dramatically different LDSOS [8]. There is a dose-dependent relationship between BHT and pulmonary DNA synthesis at low doses, but this appears to reach a maximal level long before mortality is observed, at least in BALB/c mice [16]. A similar maximization of response appears in DBA and C57 mice when lung weight is used as the index of injury [8]. Thus, it seems unlikely that lung damage is the proximal cause of death unless the indices used to quantitate injury reach maximal responses before maximal injury is achieved and therefore fail to be linearly related to dose. In the current study, the time course and extent of lung injury and repair was examined in 4 mouse strains with widely different LDses (350-1700 mg/kg). The incorporation of radiolabelled thymidine into pulmonary DNA, which has been related to the extent of the initial injury [3, 171 was used as an index of damage. Control levels of pulmonary DNA synthesis were similar in all strains except C57 mice. The slightly higher levels seen in this strain may have been related to their smaller body size. All mice received 0.5 @i thymidine, and in smaller animals this should result in a higher specific activity. Despite the wide range of doses needed to produce death, the time course of lung cell division after BHT was generally the same between strains given identical doses. This included doses of from 23% (BALB/c) tox85% (SSIn) of the LDs,-, values. In addition, a previous report in Swiss-Webster mice given 400 mg/kg BHT demonstrated a similar time course of injury and repair [7]. Some subtle differences may exist between strains, as demonstrated by the slightly more rapid repair in BALB/c and
60
ICR mice, but these differences seem unlikely to have any physiological significance. The development of pulmonary fibrosis, as reflected by lung hydroxyproline content, also failed to reveal any strain-related differences in responsiveness to BHT-induced damage. BALB/c, ICR and C57 mice had similar percentage increases in lung fibrosis which were far from the maximum achievable in these animals. Differences in absolute increases were observed, but seemed primarily due to differences in body weight and thus lung size before treatment. BHT-induced increases in pulmonary hydroxyproline were not detected in SSIn mice. This may have been related to the lower absolute dose given to these mice (300 vs. 400 mg/kg BHT), although the dose relative to the LDs,-,was the highest in this strain. Previous work has indicated a threshold dose for the development of fibrosis in mice treated with BHT [lo], and it is possible the dose given to SSIn mice did not exceed this threshold despite the obvious injury produced. Interestingly, there appears to be a maximal level of fibrosis produced by BHT in mice. Differences between mouse strains in their sensitivity to skin tumor promotion using phorbol esters have recently been related to the ability of skin tissue to undergo a sustained hyperplasiagenic response [18]. Among the mouse strains used in the present study, SSIn and DBA/2 are highly sensitive, ICR are intermediate, and BALB/c and C57BL/6 are relatively resistant to phorbol ester promotion [18]. In general, strains which exhibit higher levels of sustained hyperplasia are more susceptible to tumor promotion by phorbol esters [18]. Furthermore, a recent study demonstrated that SENCAR mice (the progenitor of SSIn) exhibited an exaggerated and persistent epidermal hyperplasia in response to tissue damage caused by ultraviolet irradiation compared to BALB/c mice [19]. One rationale for the current study was to determine if a similar relationship might hold for chemical-induced cell proliferation of internal organs. The results demonstrate, however, that the time course of repair after BHT-induced lung damage was basically similar in all strains and the minor differences which were observed do not correlate with the susceptibility of these strains to skin tumor promotion or ultraviolet-induced carcinogenesis. Thus, strains of mice with different susceptibilities to phorbol ester promotion, including the selectively bred SSIn, do not appear to differ significantly in their response to agents toxic for the lung, such as BHT. BHT has been found to both inhibit and promote tumor formation in different model systems [2]. Strain differences in the effect of BHT on urethane-induced mouse lung adenomas have been reported [20]. The mechanism of these differences has not been explored. Correlations between these differences and BHT metabolism, lung injury and overall mortality would be of interest and may provide insights into the mechanism by which BHT can function as a tumor promoter. In summary, BHT-induced lung injury is evident in all strains of mice which have been tested. The magnitude of this injury shows only minor variations between strains given equivalent doses. Thus, the overall toxicity of BHT to the different strains of mice appears not to be related to the extent of lung damage induced by this antioxidant.
61 ACKNOWLEDGEMENTS
This work was supported by NIH grants HL35689 (J.P.K.) and CA38871 (J.D.). J.P.K. is the recipient of Research Career Development Award HL01435. Thanks go to Mr. Thomas Devor for his excellent technical assistance. REFERENCES
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