- Email: [email protected]
Lung growth in mice after a single dose of butylated hydroxytoluene
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
Lung
33,309-3
19 (1975)
Growth in Mice after a Single of Butylated Hydroxytoluene WAJIH
SAHEB
AND
HANSPETER
Dose
WITSCHI
Department of Pharmacology, Faculty of Medicine, Unicersite’ de Montr&al, Montkal, QuPbec, Canada Received December 3,1974; accepted February 16,1975 Lung Growth in Mice After a Single Dose of Butylated Hydroxytoluene. W. AND WITSCHI, H. (1975).Toxicol. Appl. Pharmacol. 33,309319.
SAHEB,
In maleSwiss-Webstermice,a singleinjection of butylated hydroxytoluene (BHT) produced a dose-dependentincreasein lung weight. Histopathological changeswere well developed3 days after 500mg/kg of BHT. After 5 days, there seemedto be a proliferation of many alveolar cells,formation of giant cells,and macrophageproliferation. Lower dosesof BHT produced similar, although lessextensive changes.The histopathologic alterations wereaccompaniedby biochemicalchanges:2 daysafter BHT, there was a significantincreasein lung weight and total amountsof DNA, RNA, and protein. The changeswere dose-dependentand the smallesteffective dose was 250mg/kg of BHT. Five days after BHT, the highest dosesof BHT (500 and 1000mg!kg) produced a 1.5- to 2-fold increasein lung weight, total DNA, and protein, and a 3- to 4-fold increasein total pulmonary RNA. The incorporation of thymidine into DNA and of leucine into protein increasedfrom 2 dayson after BHT. On the other hand, the incorporation of erotic acid into total pulmonary RNA waslower in the treated animalsthan in the controls. Administration of a singledoseof BHT might offer a convenienttool to study the biochemicalchangesprecedingand/or accompanyingstimulatedcell growth in lung. In 1972,Marino and Mitchell reported that the antioxidant butylated hydroxytoluene (BHT) caused a proliferation of alveolar cells in the lungs of mice. The smallest dose required to stimulate cell growth was approximately 40 mg/kg. Proliferation of alveolar cells was most prominent 5 days after BHT. Seven days or later after a dose as high as 400 mg/kg, the lungs of the treated animals looked essentially normal again. We subsequently examined some biochemical changesproduced by the ip administration of400 mg/kg of BHT, a dosecorresponding to the LD50 in mice usedin our experiment (Witschi and Saheb, 1974). In treated animals, total lung weight increasedup to 5 days after BHT and then decreasedagain slightly toward normal values; however, even 9 days after BHT, treated animals had a significantly increased lung weight compared with controls. Total pulmonary DNA increased and remained almost twice as high in treated mice compared to controls until 9 days after BHT. The incorporation of thymidine into pulmonary DNA was found to be five to ten times higher in treated animals than in controls on Days 2-5 after BHT; later, thymidine incorporation returned Copyright 0 197.5 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
309
310
SAHEB
AND
WITSCHI
toward control values again. A stimulation of DNA synthesis was observed in lung only, but not in liver, kidneys, spleen, and the gastrointestinal tract. The experiments reported in this paper were designed to investigate in somewhat more detail several biochemical events associated with BHT-stimulated cell growth in mouse lung within the first 5 days after BHT. The smallest dose which would stimulate DNA synthesis was determined and it was examined whether increased DNA synthesis would be accompanied by alterations in the metabolism of protein and of RNA. Some of the data have been presented in preliminary form (Saheb and Witschi, 1974a, 1974b). METHODS
Young adult male Swiss-Webster mice (Bio-Breeding Co., Ottawa, Ont.) weighing 20-30 g, were housed in plastic cages with bedding in an air-conditioned room with controlled lighting. Butylated hydroxytoluene (3,5-di-tert-butyl-4 hydroxytoluene, BHT) was purchased from Sigma Chemical Company, St. Louis, MO. All radioisotopes were purchased from New England Nuclear, Waltham, Mass. BHT was dissolved in corn oil and injected ip (injection vol, 0.2 ml). All control animals received 0.2 ml of corn oil alone. After the injection, the animals had free access to food and water throughout the experiment. They were killed 1-5 days after BHT by cervical dislocation. For histology, lungs were fixed in 10% buffered formalin and paraffin embedded; 5-pm sections were stained with hematoxylin-eosin. The amounts of DNA, RNA, and protein per total lung were determined as follows; the lungs from two mice were rapidly excised, weighed individually, and homogenized in 10 ml of ice-cold water. Five milliliters of homogenate were precipitated with 2.5 ml of ice-cold 0.6 N HCIO,. The pellet was washed three times with cold 0.2 N HC104, extracted for 120 min at 37°C with 3 N KOH, and reacidified with 2.5 N HCIO,. After centrifugation, the pellet was washed once more with 0.2 N HC104 and the two supernates were combined. The RNA content was measured by the orcinol method (Volkin and Cohn, 1954). The pellet was further extracted twice with 0.5 N HC104 during 20 min each at 70-80°C and the extract was analyzed for DNA by the diphenylamine method (Burton, 1956). For the determination of protein, 2 ml of the original homogenate were precipitated with 8 ml of 0.5 N HCIO, and protein content was measured with a modified biuret procedure (Aldridge, 1957). The following chemicals were used as standards: bovine serum albumin, fraction V, RNA from yeast, and calf thymus DNA, highly polymerized (all substances purchased from Sigma Chemicals Co.). For the determination of dry lung weight and lipid content, lungs of one animal each were weighed, dried for 48 hr at lOO”C, and reweighed. The dry tissue was then extracted, under occasional agitation, for 24 hr with ether and for another 24 hr with petroleum ether, BP 30-60°C. The tissue was reweighed and the difference in weight before and after solvent extraction was assumed to represent lipid content. Incorporation of thymidine into DNA was measured as follows: the animals were injected ip with 1.5 ,uCi [2-14C]thymidine (sp act 50-60 mCi/mmol). Two hours later, the animals were killed and the specific activity of pulmonary DNA was measured as described before (Witschi, 1968). Protein synthesis was measured by the ip injection of 2 &i of DL-[1-14C]leucine (sp act 25-30 mCi/mmol); 15 min later, the animals were killed and the specific activity of total lung protein was measured as described previously
LUNG
GROWTH
AFTER
BHT
31!
(Witschi, 1972). For the evaluation of RNA synthesis, 2.5 &i of f6-14C]orotic acid hydrate (sp act 50-60 mCi/mmol) were injected into a tail vein ; 15 min later, the animals were killed and the specific activity of total pulmonary RNA was determined as described before (Witschi, 1972). Results were calculated as means &SD or &SE. Significance levels were estimated with a Student’s t-test (Snedecor and Cochran, 1967) and p values of 0.05 or less were considered to be significant.
RESULTS
In a first series of experiments, the development of histopathological lesions was followed for 5 days in mice treated with a single dose of BHT. Groups of 12 animals were given 62.5, 125, 250, and 500 mg/kg of BHT. A control group received corn oil only. On Days 1, 3, and 5 after BHT, four animals per group were killed. In control
FIG. 1. Left panel, lung from control mouse with normal alveolar structure. Middle panel, mouse lung 3 days after 500 mg/kg of BHT; alveolar septa thickened and increased cellularity. Right panel. 5 days after 500 mg/kg of BHT; occasional clusters of enlarged cells. HE, x450.
animals, no macroscopically visible lung changes were noticed. Histologically, there were in both control and treated animals minor signs of chronic respiratory infection such as occasional dense peribronchial round cell (lymphocytes) accumulation and some epithelial hyperplasia in the walls of medium sized and small airways. Besides those changes, control animals showed a normal lung morphology with well-developed respiratory airways and a fine, thin-walled alveolar network (Fig. 1).
312
SAHEB
AND
WITSCHI
In treated animals, the most extensive lesions were observed after the highest dose of BHT. Gross macroscopic changes sometimes became apparent as early as 3 days after BHT and were definite after 5 days. The lungs were enlarged and showed plum-colored patches; occasionally, an entire lobe was of uniform dark-reddish color. Histologically, some discrete changes were noticeable 1 day after BHT. The alveolar septa appeared to be somewhat swollen. Blood stasis in capillaries and small vessels and some perivascular edema were seen. However, no edema fluid or inflammatory cells were found within the alveoli. Three days after BHT, the changes had become more prominent (Fig. 1). There was a diffuse thickening of the alveolar septa, and increased cellularity. In several places appreciably enlarged cells were seen. In some cells, the nuclei were pycnotic, whereas in other cells they were enlarged. As a result of the thickening of the alveolar septa, the alveoli occasionally appeared obliterated. Blood stasis in capillaries and small vessels was quite extensive. Five days after BHT, the alveolar walls were somewhat less edematous than before, but were thickened in many areas (Fig. 1). Occasional small foci with clusters of enlarged cells were found. In other places, there were within the alveolar septa single, greatly enlarged cells whose nuclei sometimes seemed to fill almost the entire cell body. Quite a few of the enlarged cells contained two nuclei; however, mitotic figures were only rarely observed. Accumulation in the alveoli of free cells with a round, sometimes excentric, and homogeneously staining nucleus was a prominent feature in many regions of the affected lungs, whereas in other areas the lumen of the alveoli was empty. Therefore, 500 mg/kg of BHT produced, within 5 days after administration, widespread tissue damage which was interpreted to represent epithelial and macrophage proliferation. In the animals given lower doses, similar changes were observed, but in a nondose-, nonduration-related manner. Our histopathologic findings agree with the ones reported by Marino and Mitchell (1972). A chemical analysis of mouse lung after BHT was done with the following goal in mind: we wanted to examine whether the alterations observed histopathologically could be followed and quantitated with biochemical methods. For this, it was necessary to establish the time-response of biochemically measurable alterations and to find the lowest dose which would produce such changes. TABLE 1 CHEMICAL COMPOSITIONOF NORMAL MOUSE LUNG=
DNA RNA
Total wet wt 188+22mg* Total dry wt 43 f 7 mgb Water content 77.1% mg/g wet wt wz/luw 1.20 + 0.08” 6.38
Protein Lipids
0.54 + 0.06” 29.87 4 6.20’ 4.95 + 1.07b
2.87 158.88 26.33
DMale Swiss-Webster mice, 28-30 g. b Mean + SD; data obtained from 30 animals. c Mean +SD; data obtained from 30determinations on pooled lungs from two animals.
Table 1 lists the composition
of normal mouse lung under our experimental
ditions. In Figs. 2-4 we have plotted
the changes in wet lung weight,
con-
total DNA,
LUNG Total
OL
8 I
1
2
lung
8
3
GROWTH
AFTER
313
BHT Total
weight
( 4
k
01
5
Days
after
1 I
1
2
DNA
6
3
per lung
1
4
' 5
Et HT
FIG. 2. Increase in total wet weight and totalDNA per lung l-5 days after BHT. Each point represents mean &SE from four to six determinations (two pooled lungs for each) and is expressed as a percentage of corresponding controls. Statistically significant differences (p < 0.05) are indicated with an asterisk. Doses of BHT: (0) 1000 mg/kg; (0) 500 mg/kg; (A) 250 mg/kg; (0) 125 mg/kg. Total protein
per lung
Total
l~plds per lung
*
I-
5-
)-
I
2
3
4
5
Days
after
M-IT
Ftc;. 3. Increase in total protein and changes in total lipid per lung 1-5 days after BHT. Each point represents the mean &SE from four to six determinations (two pooled lungs for each) for protein or from six individual lungs for lipids, and is expressed as a percentage of corresponding controls. Statistically significant differences (p c 0.05) are indicated with an asterisk. Doses of BHT: (!?) 1000 mgjkg: (:) 500 mg/kg; (A) 250 mg/kg; (0) 125 mg/kg.
314
SAHEB
AND Total
400
-
300
-
WITSCHI RNA
per lung
T”
E 2 $ PE 200 s 5 s IOO-
01 I
2
Days
3
after
4
5
BHT
FIG. 4. Increase in total RNA per lung l-5 days after BHT. Each point represents mean +SE from four to six determinations (two pooled lungs for each) and is expressed as a percentage of corresponding control values. Statistically significant differences (p < 0.05) are indicated with an asterisk. Doses of BHT: (0) 1000 mg/kg; (0) 500 mg/kg; (A) 250 mg/kg; (0) 125 mg/kg.
protein, lipids, and RNA per lung during l-5 days after BHT (125, 250, 500, and 1000 mg/kg). Each plotted point represents the mean value of four to six determinations on the lungs pooled from two animals. For each such point, an equal number of control values had been established and the difference between experimental and control values was analyzed statistically. The results can be summarized as follows: No biochemical changes were observed 1 day after any dose of BHT. On Day 2, the lowest dose (125 mg/kg) seemed to produce a slight decrease in lung weight, DNA and RNA, and an increased lipid content per lung. Later, there were no differences between the animals treated with 125 mg/kg and their controls. The three higher doses produced clear-cut alterations in chemical lung composition; lung weight and total RNA were significantly increased over control values as early as 48 hr after BHT. On the same day, significantly more protein was found in the lungs of animals given 500 and 1000 mg/kg. Total DNA per lung was increased on Day 2 after the highest dose only. However, from Day 3 on, total lung weight, DNA, RNA, lipids, and protein were significantly higher in all animals treated with 250 mg/kg or more and the differences increased until Day 5. Therefore, the lowest dose required to produce biochemical changes was in the vicinity of 250 mg/kg of BHT. The changes in lung composition appeared to be dose-dependent, this being most clearly visible 3 days after BHT. In order to assesswhether all pulmonary constituents had increased proportionally to each other, we calculated the concentrations of RNA, protein, and lipid relative to the concentration of DNA for Day 5 after BHT (Table 2). The data show that the ratio of protein/DNA was not different between treated animals and controls. Increase in total protein was therefore strictly proportional to increase in DNA. With the two
LUNG
GROWTH
AFTER
BHT
31s
higher doses of BHT, the amount of lipid appeared to be slightly reduced relative to DNA. The most striking changes however were found in total pulmonary RNA; in treated animals, the increase in RNA greatly surpassed the increase in DNA. These data confirm the histological observations; BHT produced both hyperplasia and hypertrophy of the pulmonary cell population. TABLE OF RNA,
CONCENTRATIONS
BHT dose (m/kg) 125 250 500 I 000
PROTEIN,
AND LIPID
mg RNA/mg DNA” -- ~~~~-- --~ BHT Controls 0.35 0.62 0.94 0.91
+- 0.02 + 0.06b + 0.07b + 0.07b
0.36+ 0.43 + 0.46 + 0.44 +
0.01 0.02 0.03 0.03
2
mg OF
PER
mg protein/mg BHT
5 DAYS AFTER BHT
DNA
DNA”
mg lipids,img
Controls
18.0+OSb 25.7 + 1.1 21 .l + 1.5 22.8 -t 1.0
” Mean +SE from five to six individual determinations; lungs from two mice. h Significantly different (p < 0.05) from controls.
19.7 26.0 23.2 23.2
+ 5 I +
DNA
BHT
0.5 1 .l 2.2 1.2
4.2 4.3 3.4 3.4
each determination
+ + + +
Controls
0.2 0.5 O.lb 0.3h
5.0 4.4 4.1 4.3
+ 0.4 -c 0.2 + 0.2 Ifi 0.3
was made on the pooled
To what extent the increase in total nucleic acid and protein after BHT would be accompanied by an altered incorporation of radioactive precursors into these macromolecules was then examined. Two doses of BHT, 250 and 500 mg/kg, were used in this experiment. The data are shown in Table 3. One day after BHT no differences were TABLE INCORPORATION
OF RADIOACTIVE
PRECURSORS
INTO
3 DNA,
RNA,
AND
PROTEIN
AFTER BHT”
Days after BHT
BHT dose @x/kg)
dpm/mg DNAb
1
None 250 500
1134+ 302 1790 + 322 1603 2 374
258 + 26 199 + 21 217 i. 13
5145 + 225 5759 + 489 6022 f 357
2
None 250 500
990 k 87 1667 zk 206’ 9986 k 2286’
225 + 27 262 + 42 375 f 37’
5747 f- 426 5213 + 629 4250 f 362’
3
None 250 500
791 + 92 2561 + 271” 15360 2 2293’
154 + 13 259 L- 32’ 395 -t 55”
5373 f 337 4116 + 367’ 3747 + 36’
5
None 250 500
1846 & 261 4719 5 1966 12039 +_ 2771’
313 5 33 367 + 38 565 & 116
4740 * 237 3342 f 353’ 3543 + 327’
dpm/mg
UValues are means &SE, number of animals per group 6-S. b 1.5 uCi of f2-‘%]thvmidine iu 2 hr nrior to sacrifice. e 2 flCi of &-[I J4C]lkucine ipl5 min prior to sacrifice. d 2.5 &i of [6J4C]orotic acid hydrate iv 15 min before sacrifice. e Significantly different (p < 0.05) from control values.
protein’
dpm/mg
RNAd
316
SAHEB
AND
WITSCHI
found between controls and treated animals. From Day 2 on, incorporation of thymidine into DNA was considerably enhanced in the treated animals at both dose levels. Incorporation of leucine into total protein was significantly higher 2 days after 500 mg/kg and 3 days after both doses of BHT. The data are compatible with the conclusion that the observed net increase of DNA and protein are due to enhanced synthesis. Somewhat different results were obtained by measuring the incorporation of erotic acid into RNA. The chemical analysis had shown that RNA was the substance which accumulated most extensively in the lungs of treated mice (Fig. 4, Table 2); on the other hand, incorporation of erotic acid into RNA was decreased. The two different results do not necessarily contradict each other. It is known from other organs where rapid cell proliferation is induced that the endogenous pools of immediate RNA precursors are rapidly expanded. Injected labeled precursors are diluted to a greater degree and the specific activity of the RNA may be lower than in controls, even if the actual rate of synthesis has not changed or is even increased (Bucher and Malt, 1971). The influence of BHT on RNA metabolism will be examined in future studies.
DISCUSSION
BHT is a commonly used food additive and its toxicity has been extensively tested. To the best of our knowledge, the paper by Marino and Mitchell (1972) was the first to describe a hitherto undiscovered effect of BHT on mouse lung. We subsequently established that the cell proliferation in lung caused by BHT could be detected and measured with a biochemical approach (in vivo incorporation of thymidine into DNA) as early as 2 days after BHT (Witschi and Saheb, 1974). This prompted us to examine in somewhat more detail whether biochemical techniques might be successfully used to follow the development of the BHT-induced lung lesion. Judged by the histological appearance of the lungs of treated animals, the first subtle changes seem to appear as early as 24-48 hr after BHT administration and are most prominent after 5 days. With 250 and especially 500 mg/kg, the changes spread throughout the lungs of all animals treated. Smaller doses (62.5 and 125 mg/kg) produce focal lesions only. Biochemical signs of cell proliferation were observed 2 days after 250 mg/kg or more of BHT; 125 mg/kg failed to alter the parameters used to assesscell growth in this study. A gradual increase in total pulmonary content in nucleic acids and protein, accompanied by increased incorporation of thymidine into DNA and of leucine into protein, was observed between 2 and 5 days after BHT. There was usually a good correlation between dose and effects. It was therefore possible to follow the development of the histopathological changes with biochemical methods. However, biochemical signs of cell growth were observed only after 250 mg/kg of BHT or more. It remains to be established whether finer biochemical parameters can be found which will permit the detection of stimulated cell growth after lower doses of BHT. It is difficult to assessthe relevance of Marino and Mitchell’s (1972) and our present observations with regard to the safety of BHT as a food additive. It must be emphasized that the doses required to produce cell proliferation are several orders of magnitudes higher than the average daily intake of BHT by man. Gilbert and Golberg (1965) estimated that man might take in about 0.2 mg of BHTjkg body wt daily. In a chronic feed-
LUNG
GROWTH
AFTER
BHT
317
ing study in rats the no-effect level of BHT was established at 25 mg/kg. Unless lung damage can be demonstrated after much lower doses than the ones used in this study and also in species other than mouse, there seems to be no need for undue concern. However, BHT would seem to be an excellent experimental tool to study stimulated cell growth in lung. The biochemical and morphological events associated with stimulated cell growth have been studied extensively in several organs. One of the most thoroughly examined models is the initiation of cell growth in mouse and rat salivary glands, where DNA synthesis increases 20 hr after administration of 1 mmol/kg of isoproterenol. Between 24 and 30 hr, there is maximal DNA synthesis, followed by mitosis in up to 80 “j;; of all cells (Baserga, 1968). Essentially a similar time-sequence of stimulated DNA synthesis is observed in regenerating liver (Bucher and Malt, 1971) and lead-induced proliferation of kidney tubule cells (Choie and Richter, 1974). In BHT-stimulated mouse lung. onset and especially peak activity of induced DNA synthesis occur much later. It also takes several days before DNA synthesis returns to basal levels again (Witschi and Saheb, 1974). The reasons for this delayed onset and prolonged duration of the action of BHT are not clear. The broad peak ofincreased DNA synthesis, stretched over several days, might suggest that the many different cell types present in the pulmonary parenchyma respond at different times to BHT. So far, we do not know for sure what pulmonary cell type(s) respond(s) to BHT or whether some of the cells induced to divide do so more than once. Preliminary observations suggest that type II alveolar cells increase substantially in size after BHT (M. G. Cbte, personal communication). This could mean that BHT produces a proliferation of type II alveolar cells, among others. Proliferation of these cells is a well-known response of lung tissue to toxic injury. Type II alveolar cells replace damaged type I alveolar cells (Adamson and Bowden, 1974). They have been observed to proliferate in lungs damaged by oxygen (Kapanci et al., 1969), ozone (Stephens et al., 1974), nitrogen dioxide (Stephens et al., 1972), paraquat (Vijeyaratnam and Corrin, 1971) nickel carbonyl (Hackett and Sunderman, 1967), and also in chronic pulmonary oedema (Ortega et al., 1970). A variety of drugs such as the anorexigenic agent chlorphentermine (Smith et al., 1973), the inhibitor of cholesterol biosynthesis triparanol (Liillman et al., 1973), the cytostatic agent busulphan (Littler et al., 1969), the antihistaminic agent chlorcyclizine (Hruban et al., 1973), and urethan (Kauffman, 1972). produce type II alveolar cell growth. It has recently been pointed out that proliferation of these cells not only serves to repair chemically damaged lung tissue, but actually helps to strengthen the biochemical defense mechanisms of lung tissue against many potential forms of damage, including oxidant-induced injury (Cross, 1974), If it could be shown in further studies that BHT produces a substantial proliferation of type 11 alveolar cells, then BHT might become a very useful tool to study the biochemical events which trigger and/or accompany the proliferation of these important lung cells.
ACKNOWLEDGMENTS This work was supportedby the Medical ResearchCouncil (MRC Group in Drug Toxicology). Excellent technicalhelp wasprovided by Miss Monique Morisset and Miss Francine Boursier.
318
SAHEB
AND
WITSCHI
REFERENCES I. Y. R. AND BOWDEN, D. H. (1974). The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab. Invest.
ADAMSON,
30, 35-42.
W. N. (1957).Liver and brain mitochondria. Biochem. J. 67,423-431. R. (1968). Cellular proliferation in the isoproterenol-stimulatedsalivary gland. In Proliferation and Spread of Neoplastic Cells. Annual Symposiumon FundamentalCancer Research,pp. 261-267.MD Anderson Hospitaland Tumor Institute, TexasMedical Center, Houston. BUCHER, N. L. R. AND MALT, R. A. (1971).Regeneration of Liver and Kidney. New England Journal of Medicine Medical ProgressSeries.Little, Brown and Co., Boston. BURTON, K. (1956).A study of the conditionsand mechanismof the diphenylaminereaction for the calorimetric estimationof deoxyribonucleicacid. Biochem. J. 62, 315-323. CHOIE, D. D. AND RICHTER, G. W. (1974).Cell proliferation in mousekidney inducedby lead. I. Synthesisof deoxyribonucleicacid. Lab. Invest. 30, 647-651. CROSS, C. E. (1974).The granular type II pneumocyteand lung antioxidant defense.Ann. Znt Med. 80, 409411. GILBERT, D. AND GOLBERC, L. (1965). Liver Response tests. III. Liver enlargementand stimulation of microsomalenzyme processingactivity. Food Cosmet. Toxicol. 3, 417-432. HACKETT, R. L. AND SUNDERMAN, F. W., Jr. (1967).Acute pathological reactions to administration of nickel carbonyl. Arch. Environ. Health 14,604-613. HRUBAN, Z., SLESERS, A. AND ASCHENBRENNER, I. (1973). Pulmonary intraalveolar histiocytosisinducedby drugs. Toxicol. Appl. Pharmacol. 26,72-85. KAPANCI, Y., WEIBEL, E. R., KAPLAN, H. P., AND ROBINSON, F. R. (1969).Pathogenesis and reversibility of the pulmonary lesionsof oxygen toxicity in monkeys. II. Ultrastructural and morphometric studies.Lab. Invest. 20, 101-108. KAUFFMAN, S. L. (1972).Alterations in cell proliferation in mouse lung following urethane exposure.III. Effects of chronic exposureon type 2 alveolar epithelialcells.Amer. J. Pathol. ALDRIDGE, BASERGA,
68,317-325. LITTLER,
W. A.,
KAY,
J. M.,
HASLETON,
P. S.AND
HEATH,
D. (1969).Busulphan
lung.
Thorax
24,639-655. H., L~~LLMANN-RAUCH, R. AND REIL, G. H. (1973).A comparativeultrastructural study of the effects of chlorphentermineand triparanol in rat lung and adrenal gland.
LULLMANN,
Virchows Arch. Abt. B. Zellpathol.
12,91-103.
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. ORTEGA, P., UHLEY, H. N., LEEDS, S. E., FRIEDMAN, M. AND SAMPSON, J. J. (1970).Serialelectron and light microscopic studieson the dog lung in chronic experimental pulmonary edema.Amer. J. Pathol. 60, 57-73. SAHEB, W. AND WITSCHI, H. P. (1974a).Toxicity of butylated hydroxytoluene (BHT) in mouse lung. Proc. Can. Fed. Biol. Sot. 17, 52. SAHEB, W. AND WITSCHI, H. P. (1974b).Stimulation of cellgrowth in mouselung by butylated hydroxytoluene. Pharmacologist 16,230. SMITH, P., HEATH, D. AND HASLETON, P. S. (1973).Electron microscopyof chlorphentermine lung. Thorax 28,559-568. SNEDECOR, G. W. AND COCHRAN, W. G. (1967).The comparisonof two samples.In Statistical Methods, edition 6, pp. 114-l 15. The Iowa State University Press,Ames, Iowa. STEPHENS, R. J., FREEMAN, G. AND EVANS, M. J. (1972).Early responseof lungsto low levelsof nitrogen dioxide : light and electron microscopy. Arch. Environ. Health 24, 16&l 79. STEPHENS, R. J., SLOAN, M. F., EVANS, M. J. AND FREEMAN, G. (1974).Early responseof lung to low levelsof ozone. Amer. J. Pathol. 74, 31-58. VIJEYARATNAM, G. S. AND CORRIN, B. (1971).Experimentalparaquatpoisoning:a histological and electron-optical study of the changesin the lung. J. Pathol. 103, 123-129. MARINO,
LUNG GROWTH AFTER BHT VOLKIN, E. AND COHN, W. E. 287-303. WITSCHI,
(1954).
319
Estimation of nucleic acids. Meth. Biochem. Anal. 1.
H. P. (1968).Inhibition of deoxyribonucleic acid synthesisin regeneratingrat liver by beryllium. Lab. Znuest.19, 67-70. WITSCHI,H. P. (1972).A comparative study of in vivo RNA and protein synthesisin rat liver and lung. Cancer Res. 32, 1686-1694. WITSCHI,H. P. ANDSAHEB,W. (1974).Stimulation of DNA synthesisin mouselung following intraperitoneal injection of butylated hydroxytoluene. Proc. Sot. Esp. Biol. Mea’. 147. 690-693.
AND
APPLIED
PHARMACOLOGY
Lung
33,309-3
19 (1975)
Growth in Mice after a Single of Butylated Hydroxytoluene WAJIH
SAHEB
AND
HANSPETER
Dose
WITSCHI
Department of Pharmacology, Faculty of Medicine, Unicersite’ de Montr&al, Montkal, QuPbec, Canada Received December 3,1974; accepted February 16,1975 Lung Growth in Mice After a Single Dose of Butylated Hydroxytoluene. W. AND WITSCHI, H. (1975).Toxicol. Appl. Pharmacol. 33,309319.
SAHEB,
In maleSwiss-Webstermice,a singleinjection of butylated hydroxytoluene (BHT) produced a dose-dependentincreasein lung weight. Histopathological changeswere well developed3 days after 500mg/kg of BHT. After 5 days, there seemedto be a proliferation of many alveolar cells,formation of giant cells,and macrophageproliferation. Lower dosesof BHT produced similar, although lessextensive changes.The histopathologic alterations wereaccompaniedby biochemicalchanges:2 daysafter BHT, there was a significantincreasein lung weight and total amountsof DNA, RNA, and protein. The changeswere dose-dependentand the smallesteffective dose was 250mg/kg of BHT. Five days after BHT, the highest dosesof BHT (500 and 1000mg!kg) produced a 1.5- to 2-fold increasein lung weight, total DNA, and protein, and a 3- to 4-fold increasein total pulmonary RNA. The incorporation of thymidine into DNA and of leucine into protein increasedfrom 2 dayson after BHT. On the other hand, the incorporation of erotic acid into total pulmonary RNA waslower in the treated animalsthan in the controls. Administration of a singledoseof BHT might offer a convenienttool to study the biochemicalchangesprecedingand/or accompanyingstimulatedcell growth in lung. In 1972,Marino and Mitchell reported that the antioxidant butylated hydroxytoluene (BHT) caused a proliferation of alveolar cells in the lungs of mice. The smallest dose required to stimulate cell growth was approximately 40 mg/kg. Proliferation of alveolar cells was most prominent 5 days after BHT. Seven days or later after a dose as high as 400 mg/kg, the lungs of the treated animals looked essentially normal again. We subsequently examined some biochemical changesproduced by the ip administration of400 mg/kg of BHT, a dosecorresponding to the LD50 in mice usedin our experiment (Witschi and Saheb, 1974). In treated animals, total lung weight increasedup to 5 days after BHT and then decreasedagain slightly toward normal values; however, even 9 days after BHT, treated animals had a significantly increased lung weight compared with controls. Total pulmonary DNA increased and remained almost twice as high in treated mice compared to controls until 9 days after BHT. The incorporation of thymidine into pulmonary DNA was found to be five to ten times higher in treated animals than in controls on Days 2-5 after BHT; later, thymidine incorporation returned Copyright 0 197.5 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
309
310
SAHEB
AND
WITSCHI
toward control values again. A stimulation of DNA synthesis was observed in lung only, but not in liver, kidneys, spleen, and the gastrointestinal tract. The experiments reported in this paper were designed to investigate in somewhat more detail several biochemical events associated with BHT-stimulated cell growth in mouse lung within the first 5 days after BHT. The smallest dose which would stimulate DNA synthesis was determined and it was examined whether increased DNA synthesis would be accompanied by alterations in the metabolism of protein and of RNA. Some of the data have been presented in preliminary form (Saheb and Witschi, 1974a, 1974b). METHODS
Young adult male Swiss-Webster mice (Bio-Breeding Co., Ottawa, Ont.) weighing 20-30 g, were housed in plastic cages with bedding in an air-conditioned room with controlled lighting. Butylated hydroxytoluene (3,5-di-tert-butyl-4 hydroxytoluene, BHT) was purchased from Sigma Chemical Company, St. Louis, MO. All radioisotopes were purchased from New England Nuclear, Waltham, Mass. BHT was dissolved in corn oil and injected ip (injection vol, 0.2 ml). All control animals received 0.2 ml of corn oil alone. After the injection, the animals had free access to food and water throughout the experiment. They were killed 1-5 days after BHT by cervical dislocation. For histology, lungs were fixed in 10% buffered formalin and paraffin embedded; 5-pm sections were stained with hematoxylin-eosin. The amounts of DNA, RNA, and protein per total lung were determined as follows; the lungs from two mice were rapidly excised, weighed individually, and homogenized in 10 ml of ice-cold water. Five milliliters of homogenate were precipitated with 2.5 ml of ice-cold 0.6 N HCIO,. The pellet was washed three times with cold 0.2 N HC104, extracted for 120 min at 37°C with 3 N KOH, and reacidified with 2.5 N HCIO,. After centrifugation, the pellet was washed once more with 0.2 N HC104 and the two supernates were combined. The RNA content was measured by the orcinol method (Volkin and Cohn, 1954). The pellet was further extracted twice with 0.5 N HC104 during 20 min each at 70-80°C and the extract was analyzed for DNA by the diphenylamine method (Burton, 1956). For the determination of protein, 2 ml of the original homogenate were precipitated with 8 ml of 0.5 N HCIO, and protein content was measured with a modified biuret procedure (Aldridge, 1957). The following chemicals were used as standards: bovine serum albumin, fraction V, RNA from yeast, and calf thymus DNA, highly polymerized (all substances purchased from Sigma Chemicals Co.). For the determination of dry lung weight and lipid content, lungs of one animal each were weighed, dried for 48 hr at lOO”C, and reweighed. The dry tissue was then extracted, under occasional agitation, for 24 hr with ether and for another 24 hr with petroleum ether, BP 30-60°C. The tissue was reweighed and the difference in weight before and after solvent extraction was assumed to represent lipid content. Incorporation of thymidine into DNA was measured as follows: the animals were injected ip with 1.5 ,uCi [2-14C]thymidine (sp act 50-60 mCi/mmol). Two hours later, the animals were killed and the specific activity of pulmonary DNA was measured as described before (Witschi, 1968). Protein synthesis was measured by the ip injection of 2 &i of DL-[1-14C]leucine (sp act 25-30 mCi/mmol); 15 min later, the animals were killed and the specific activity of total lung protein was measured as described previously
LUNG
GROWTH
AFTER
BHT
31!
(Witschi, 1972). For the evaluation of RNA synthesis, 2.5 &i of f6-14C]orotic acid hydrate (sp act 50-60 mCi/mmol) were injected into a tail vein ; 15 min later, the animals were killed and the specific activity of total pulmonary RNA was determined as described before (Witschi, 1972). Results were calculated as means &SD or &SE. Significance levels were estimated with a Student’s t-test (Snedecor and Cochran, 1967) and p values of 0.05 or less were considered to be significant.
RESULTS
In a first series of experiments, the development of histopathological lesions was followed for 5 days in mice treated with a single dose of BHT. Groups of 12 animals were given 62.5, 125, 250, and 500 mg/kg of BHT. A control group received corn oil only. On Days 1, 3, and 5 after BHT, four animals per group were killed. In control
FIG. 1. Left panel, lung from control mouse with normal alveolar structure. Middle panel, mouse lung 3 days after 500 mg/kg of BHT; alveolar septa thickened and increased cellularity. Right panel. 5 days after 500 mg/kg of BHT; occasional clusters of enlarged cells. HE, x450.
animals, no macroscopically visible lung changes were noticed. Histologically, there were in both control and treated animals minor signs of chronic respiratory infection such as occasional dense peribronchial round cell (lymphocytes) accumulation and some epithelial hyperplasia in the walls of medium sized and small airways. Besides those changes, control animals showed a normal lung morphology with well-developed respiratory airways and a fine, thin-walled alveolar network (Fig. 1).
312
SAHEB
AND
WITSCHI
In treated animals, the most extensive lesions were observed after the highest dose of BHT. Gross macroscopic changes sometimes became apparent as early as 3 days after BHT and were definite after 5 days. The lungs were enlarged and showed plum-colored patches; occasionally, an entire lobe was of uniform dark-reddish color. Histologically, some discrete changes were noticeable 1 day after BHT. The alveolar septa appeared to be somewhat swollen. Blood stasis in capillaries and small vessels and some perivascular edema were seen. However, no edema fluid or inflammatory cells were found within the alveoli. Three days after BHT, the changes had become more prominent (Fig. 1). There was a diffuse thickening of the alveolar septa, and increased cellularity. In several places appreciably enlarged cells were seen. In some cells, the nuclei were pycnotic, whereas in other cells they were enlarged. As a result of the thickening of the alveolar septa, the alveoli occasionally appeared obliterated. Blood stasis in capillaries and small vessels was quite extensive. Five days after BHT, the alveolar walls were somewhat less edematous than before, but were thickened in many areas (Fig. 1). Occasional small foci with clusters of enlarged cells were found. In other places, there were within the alveolar septa single, greatly enlarged cells whose nuclei sometimes seemed to fill almost the entire cell body. Quite a few of the enlarged cells contained two nuclei; however, mitotic figures were only rarely observed. Accumulation in the alveoli of free cells with a round, sometimes excentric, and homogeneously staining nucleus was a prominent feature in many regions of the affected lungs, whereas in other areas the lumen of the alveoli was empty. Therefore, 500 mg/kg of BHT produced, within 5 days after administration, widespread tissue damage which was interpreted to represent epithelial and macrophage proliferation. In the animals given lower doses, similar changes were observed, but in a nondose-, nonduration-related manner. Our histopathologic findings agree with the ones reported by Marino and Mitchell (1972). A chemical analysis of mouse lung after BHT was done with the following goal in mind: we wanted to examine whether the alterations observed histopathologically could be followed and quantitated with biochemical methods. For this, it was necessary to establish the time-response of biochemically measurable alterations and to find the lowest dose which would produce such changes. TABLE 1 CHEMICAL COMPOSITIONOF NORMAL MOUSE LUNG=
DNA RNA
Total wet wt 188+22mg* Total dry wt 43 f 7 mgb Water content 77.1% mg/g wet wt wz/luw 1.20 + 0.08” 6.38
Protein Lipids
0.54 + 0.06” 29.87 4 6.20’ 4.95 + 1.07b
2.87 158.88 26.33
DMale Swiss-Webster mice, 28-30 g. b Mean + SD; data obtained from 30 animals. c Mean +SD; data obtained from 30determinations on pooled lungs from two animals.
Table 1 lists the composition
of normal mouse lung under our experimental
ditions. In Figs. 2-4 we have plotted
the changes in wet lung weight,
con-
total DNA,
LUNG Total
OL
8 I
1
2
lung
8
3
GROWTH
AFTER
313
BHT Total
weight
( 4
k
01
5
Days
after
1 I
1
2
DNA
6
3
per lung
1
4
' 5
Et HT
FIG. 2. Increase in total wet weight and totalDNA per lung l-5 days after BHT. Each point represents mean &SE from four to six determinations (two pooled lungs for each) and is expressed as a percentage of corresponding controls. Statistically significant differences (p < 0.05) are indicated with an asterisk. Doses of BHT: (0) 1000 mg/kg; (0) 500 mg/kg; (A) 250 mg/kg; (0) 125 mg/kg. Total protein
per lung
Total
l~plds per lung
*
I-
5-
)-
I
2
3
4
5
Days
after
M-IT
Ftc;. 3. Increase in total protein and changes in total lipid per lung 1-5 days after BHT. Each point represents the mean &SE from four to six determinations (two pooled lungs for each) for protein or from six individual lungs for lipids, and is expressed as a percentage of corresponding controls. Statistically significant differences (p c 0.05) are indicated with an asterisk. Doses of BHT: (!?) 1000 mgjkg: (:) 500 mg/kg; (A) 250 mg/kg; (0) 125 mg/kg.
314
SAHEB
AND Total
400
-
300
-
WITSCHI RNA
per lung
T”
E 2 $ PE 200 s 5 s IOO-
01 I
2
Days
3
after
4
5
BHT
FIG. 4. Increase in total RNA per lung l-5 days after BHT. Each point represents mean +SE from four to six determinations (two pooled lungs for each) and is expressed as a percentage of corresponding control values. Statistically significant differences (p < 0.05) are indicated with an asterisk. Doses of BHT: (0) 1000 mg/kg; (0) 500 mg/kg; (A) 250 mg/kg; (0) 125 mg/kg.
protein, lipids, and RNA per lung during l-5 days after BHT (125, 250, 500, and 1000 mg/kg). Each plotted point represents the mean value of four to six determinations on the lungs pooled from two animals. For each such point, an equal number of control values had been established and the difference between experimental and control values was analyzed statistically. The results can be summarized as follows: No biochemical changes were observed 1 day after any dose of BHT. On Day 2, the lowest dose (125 mg/kg) seemed to produce a slight decrease in lung weight, DNA and RNA, and an increased lipid content per lung. Later, there were no differences between the animals treated with 125 mg/kg and their controls. The three higher doses produced clear-cut alterations in chemical lung composition; lung weight and total RNA were significantly increased over control values as early as 48 hr after BHT. On the same day, significantly more protein was found in the lungs of animals given 500 and 1000 mg/kg. Total DNA per lung was increased on Day 2 after the highest dose only. However, from Day 3 on, total lung weight, DNA, RNA, lipids, and protein were significantly higher in all animals treated with 250 mg/kg or more and the differences increased until Day 5. Therefore, the lowest dose required to produce biochemical changes was in the vicinity of 250 mg/kg of BHT. The changes in lung composition appeared to be dose-dependent, this being most clearly visible 3 days after BHT. In order to assesswhether all pulmonary constituents had increased proportionally to each other, we calculated the concentrations of RNA, protein, and lipid relative to the concentration of DNA for Day 5 after BHT (Table 2). The data show that the ratio of protein/DNA was not different between treated animals and controls. Increase in total protein was therefore strictly proportional to increase in DNA. With the two
LUNG
GROWTH
AFTER
BHT
31s
higher doses of BHT, the amount of lipid appeared to be slightly reduced relative to DNA. The most striking changes however were found in total pulmonary RNA; in treated animals, the increase in RNA greatly surpassed the increase in DNA. These data confirm the histological observations; BHT produced both hyperplasia and hypertrophy of the pulmonary cell population. TABLE OF RNA,
CONCENTRATIONS
BHT dose (m/kg) 125 250 500 I 000
PROTEIN,
AND LIPID
mg RNA/mg DNA” -- ~~~~-- --~ BHT Controls 0.35 0.62 0.94 0.91
+- 0.02 + 0.06b + 0.07b + 0.07b
0.36+ 0.43 + 0.46 + 0.44 +
0.01 0.02 0.03 0.03
2
mg OF
PER
mg protein/mg BHT
5 DAYS AFTER BHT
DNA
DNA”
mg lipids,img
Controls
18.0+OSb 25.7 + 1.1 21 .l + 1.5 22.8 -t 1.0
” Mean +SE from five to six individual determinations; lungs from two mice. h Significantly different (p < 0.05) from controls.
19.7 26.0 23.2 23.2
+ 5 I +
DNA
BHT
0.5 1 .l 2.2 1.2
4.2 4.3 3.4 3.4
each determination
+ + + +
Controls
0.2 0.5 O.lb 0.3h
5.0 4.4 4.1 4.3
+ 0.4 -c 0.2 + 0.2 Ifi 0.3
was made on the pooled
To what extent the increase in total nucleic acid and protein after BHT would be accompanied by an altered incorporation of radioactive precursors into these macromolecules was then examined. Two doses of BHT, 250 and 500 mg/kg, were used in this experiment. The data are shown in Table 3. One day after BHT no differences were TABLE INCORPORATION
OF RADIOACTIVE
PRECURSORS
INTO
3 DNA,
RNA,
AND
PROTEIN
AFTER BHT”
Days after BHT
BHT dose @x/kg)
dpm/mg DNAb
1
None 250 500
1134+ 302 1790 + 322 1603 2 374
258 + 26 199 + 21 217 i. 13
5145 + 225 5759 + 489 6022 f 357
2
None 250 500
990 k 87 1667 zk 206’ 9986 k 2286’
225 + 27 262 + 42 375 f 37’
5747 f- 426 5213 + 629 4250 f 362’
3
None 250 500
791 + 92 2561 + 271” 15360 2 2293’
154 + 13 259 L- 32’ 395 -t 55”
5373 f 337 4116 + 367’ 3747 + 36’
5
None 250 500
1846 & 261 4719 5 1966 12039 +_ 2771’
313 5 33 367 + 38 565 & 116
4740 * 237 3342 f 353’ 3543 + 327’
dpm/mg
UValues are means &SE, number of animals per group 6-S. b 1.5 uCi of f2-‘%]thvmidine iu 2 hr nrior to sacrifice. e 2 flCi of &-[I J4C]lkucine ipl5 min prior to sacrifice. d 2.5 &i of [6J4C]orotic acid hydrate iv 15 min before sacrifice. e Significantly different (p < 0.05) from control values.
protein’
dpm/mg
RNAd
316
SAHEB
AND
WITSCHI
found between controls and treated animals. From Day 2 on, incorporation of thymidine into DNA was considerably enhanced in the treated animals at both dose levels. Incorporation of leucine into total protein was significantly higher 2 days after 500 mg/kg and 3 days after both doses of BHT. The data are compatible with the conclusion that the observed net increase of DNA and protein are due to enhanced synthesis. Somewhat different results were obtained by measuring the incorporation of erotic acid into RNA. The chemical analysis had shown that RNA was the substance which accumulated most extensively in the lungs of treated mice (Fig. 4, Table 2); on the other hand, incorporation of erotic acid into RNA was decreased. The two different results do not necessarily contradict each other. It is known from other organs where rapid cell proliferation is induced that the endogenous pools of immediate RNA precursors are rapidly expanded. Injected labeled precursors are diluted to a greater degree and the specific activity of the RNA may be lower than in controls, even if the actual rate of synthesis has not changed or is even increased (Bucher and Malt, 1971). The influence of BHT on RNA metabolism will be examined in future studies.
DISCUSSION
BHT is a commonly used food additive and its toxicity has been extensively tested. To the best of our knowledge, the paper by Marino and Mitchell (1972) was the first to describe a hitherto undiscovered effect of BHT on mouse lung. We subsequently established that the cell proliferation in lung caused by BHT could be detected and measured with a biochemical approach (in vivo incorporation of thymidine into DNA) as early as 2 days after BHT (Witschi and Saheb, 1974). This prompted us to examine in somewhat more detail whether biochemical techniques might be successfully used to follow the development of the BHT-induced lung lesion. Judged by the histological appearance of the lungs of treated animals, the first subtle changes seem to appear as early as 24-48 hr after BHT administration and are most prominent after 5 days. With 250 and especially 500 mg/kg, the changes spread throughout the lungs of all animals treated. Smaller doses (62.5 and 125 mg/kg) produce focal lesions only. Biochemical signs of cell proliferation were observed 2 days after 250 mg/kg or more of BHT; 125 mg/kg failed to alter the parameters used to assesscell growth in this study. A gradual increase in total pulmonary content in nucleic acids and protein, accompanied by increased incorporation of thymidine into DNA and of leucine into protein, was observed between 2 and 5 days after BHT. There was usually a good correlation between dose and effects. It was therefore possible to follow the development of the histopathological changes with biochemical methods. However, biochemical signs of cell growth were observed only after 250 mg/kg of BHT or more. It remains to be established whether finer biochemical parameters can be found which will permit the detection of stimulated cell growth after lower doses of BHT. It is difficult to assessthe relevance of Marino and Mitchell’s (1972) and our present observations with regard to the safety of BHT as a food additive. It must be emphasized that the doses required to produce cell proliferation are several orders of magnitudes higher than the average daily intake of BHT by man. Gilbert and Golberg (1965) estimated that man might take in about 0.2 mg of BHTjkg body wt daily. In a chronic feed-
LUNG
GROWTH
AFTER
BHT
317
ing study in rats the no-effect level of BHT was established at 25 mg/kg. Unless lung damage can be demonstrated after much lower doses than the ones used in this study and also in species other than mouse, there seems to be no need for undue concern. However, BHT would seem to be an excellent experimental tool to study stimulated cell growth in lung. The biochemical and morphological events associated with stimulated cell growth have been studied extensively in several organs. One of the most thoroughly examined models is the initiation of cell growth in mouse and rat salivary glands, where DNA synthesis increases 20 hr after administration of 1 mmol/kg of isoproterenol. Between 24 and 30 hr, there is maximal DNA synthesis, followed by mitosis in up to 80 “j;; of all cells (Baserga, 1968). Essentially a similar time-sequence of stimulated DNA synthesis is observed in regenerating liver (Bucher and Malt, 1971) and lead-induced proliferation of kidney tubule cells (Choie and Richter, 1974). In BHT-stimulated mouse lung. onset and especially peak activity of induced DNA synthesis occur much later. It also takes several days before DNA synthesis returns to basal levels again (Witschi and Saheb, 1974). The reasons for this delayed onset and prolonged duration of the action of BHT are not clear. The broad peak ofincreased DNA synthesis, stretched over several days, might suggest that the many different cell types present in the pulmonary parenchyma respond at different times to BHT. So far, we do not know for sure what pulmonary cell type(s) respond(s) to BHT or whether some of the cells induced to divide do so more than once. Preliminary observations suggest that type II alveolar cells increase substantially in size after BHT (M. G. Cbte, personal communication). This could mean that BHT produces a proliferation of type II alveolar cells, among others. Proliferation of these cells is a well-known response of lung tissue to toxic injury. Type II alveolar cells replace damaged type I alveolar cells (Adamson and Bowden, 1974). They have been observed to proliferate in lungs damaged by oxygen (Kapanci et al., 1969), ozone (Stephens et al., 1974), nitrogen dioxide (Stephens et al., 1972), paraquat (Vijeyaratnam and Corrin, 1971) nickel carbonyl (Hackett and Sunderman, 1967), and also in chronic pulmonary oedema (Ortega et al., 1970). A variety of drugs such as the anorexigenic agent chlorphentermine (Smith et al., 1973), the inhibitor of cholesterol biosynthesis triparanol (Liillman et al., 1973), the cytostatic agent busulphan (Littler et al., 1969), the antihistaminic agent chlorcyclizine (Hruban et al., 1973), and urethan (Kauffman, 1972). produce type II alveolar cell growth. It has recently been pointed out that proliferation of these cells not only serves to repair chemically damaged lung tissue, but actually helps to strengthen the biochemical defense mechanisms of lung tissue against many potential forms of damage, including oxidant-induced injury (Cross, 1974), If it could be shown in further studies that BHT produces a substantial proliferation of type 11 alveolar cells, then BHT might become a very useful tool to study the biochemical events which trigger and/or accompany the proliferation of these important lung cells.
ACKNOWLEDGMENTS This work was supportedby the Medical ResearchCouncil (MRC Group in Drug Toxicology). Excellent technicalhelp wasprovided by Miss Monique Morisset and Miss Francine Boursier.
318
SAHEB
AND
WITSCHI
REFERENCES I. Y. R. AND BOWDEN, D. H. (1974). The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab. Invest.
ADAMSON,
30, 35-42.
W. N. (1957).Liver and brain mitochondria. Biochem. J. 67,423-431. R. (1968). Cellular proliferation in the isoproterenol-stimulatedsalivary gland. In Proliferation and Spread of Neoplastic Cells. Annual Symposiumon FundamentalCancer Research,pp. 261-267.MD Anderson Hospitaland Tumor Institute, TexasMedical Center, Houston. BUCHER, N. L. R. AND MALT, R. A. (1971).Regeneration of Liver and Kidney. New England Journal of Medicine Medical ProgressSeries.Little, Brown and Co., Boston. BURTON, K. (1956).A study of the conditionsand mechanismof the diphenylaminereaction for the calorimetric estimationof deoxyribonucleicacid. Biochem. J. 62, 315-323. CHOIE, D. D. AND RICHTER, G. W. (1974).Cell proliferation in mousekidney inducedby lead. I. Synthesisof deoxyribonucleicacid. Lab. Invest. 30, 647-651. CROSS, C. E. (1974).The granular type II pneumocyteand lung antioxidant defense.Ann. Znt Med. 80, 409411. GILBERT, D. AND GOLBERC, L. (1965). Liver Response tests. III. Liver enlargementand stimulation of microsomalenzyme processingactivity. Food Cosmet. Toxicol. 3, 417-432. HACKETT, R. L. AND SUNDERMAN, F. W., Jr. (1967).Acute pathological reactions to administration of nickel carbonyl. Arch. Environ. Health 14,604-613. HRUBAN, Z., SLESERS, A. AND ASCHENBRENNER, I. (1973). Pulmonary intraalveolar histiocytosisinducedby drugs. Toxicol. Appl. Pharmacol. 26,72-85. KAPANCI, Y., WEIBEL, E. R., KAPLAN, H. P., AND ROBINSON, F. R. (1969).Pathogenesis and reversibility of the pulmonary lesionsof oxygen toxicity in monkeys. II. Ultrastructural and morphometric studies.Lab. Invest. 20, 101-108. KAUFFMAN, S. L. (1972).Alterations in cell proliferation in mouse lung following urethane exposure.III. Effects of chronic exposureon type 2 alveolar epithelialcells.Amer. J. Pathol. ALDRIDGE, BASERGA,
68,317-325. LITTLER,
W. A.,
KAY,
J. M.,
HASLETON,
P. S.AND
HEATH,
D. (1969).Busulphan
lung.
Thorax
24,639-655. H., L~~LLMANN-RAUCH, R. AND REIL, G. H. (1973).A comparativeultrastructural study of the effects of chlorphentermineand triparanol in rat lung and adrenal gland.
LULLMANN,
Virchows Arch. Abt. B. Zellpathol.
12,91-103.
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. ORTEGA, P., UHLEY, H. N., LEEDS, S. E., FRIEDMAN, M. AND SAMPSON, J. J. (1970).Serialelectron and light microscopic studieson the dog lung in chronic experimental pulmonary edema.Amer. J. Pathol. 60, 57-73. SAHEB, W. AND WITSCHI, H. P. (1974a).Toxicity of butylated hydroxytoluene (BHT) in mouse lung. Proc. Can. Fed. Biol. Sot. 17, 52. SAHEB, W. AND WITSCHI, H. P. (1974b).Stimulation of cellgrowth in mouselung by butylated hydroxytoluene. Pharmacologist 16,230. SMITH, P., HEATH, D. AND HASLETON, P. S. (1973).Electron microscopyof chlorphentermine lung. Thorax 28,559-568. SNEDECOR, G. W. AND COCHRAN, W. G. (1967).The comparisonof two samples.In Statistical Methods, edition 6, pp. 114-l 15. The Iowa State University Press,Ames, Iowa. STEPHENS, R. J., FREEMAN, G. AND EVANS, M. J. (1972).Early responseof lungsto low levelsof nitrogen dioxide : light and electron microscopy. Arch. Environ. Health 24, 16&l 79. STEPHENS, R. J., SLOAN, M. F., EVANS, M. J. AND FREEMAN, G. (1974).Early responseof lung to low levelsof ozone. Amer. J. Pathol. 74, 31-58. VIJEYARATNAM, G. S. AND CORRIN, B. (1971).Experimentalparaquatpoisoning:a histological and electron-optical study of the changesin the lung. J. Pathol. 103, 123-129. MARINO,
LUNG GROWTH AFTER BHT VOLKIN, E. AND COHN, W. E. 287-303. WITSCHI,
(1954).
319
Estimation of nucleic acids. Meth. Biochem. Anal. 1.
H. P. (1968).Inhibition of deoxyribonucleic acid synthesisin regeneratingrat liver by beryllium. Lab. Znuest.19, 67-70. WITSCHI,H. P. (1972).A comparative study of in vivo RNA and protein synthesisin rat liver and lung. Cancer Res. 32, 1686-1694. WITSCHI,H. P. ANDSAHEB,W. (1974).Stimulation of DNA synthesisin mouselung following intraperitoneal injection of butylated hydroxytoluene. Proc. Sot. Esp. Biol. Mea’. 147. 690-693.