Environmental health science in the city by the Bay.

The present investigation shows the effect of vitamin A deficiency on the initiation and postinitiation phases of benzo(a)pyrene-induced lung carcinogenesis in male Wistar rats. Lung tumours were induced by giving three intratracheal instillations, one week apart, of 10 mg benzo(a)pyrene per instillation. Maximum tumour incidence (100%) and tumour burden per rat was found in rats which were kept on vitamin A deficient diet for 4 weeks prior to the first administration and 8 weeks after the last administration of benzo(a)pyrene. Rats in which vitamin A deficiency was terminated after the last administration of the carcinogen had 83% tumour incidence, whereas corresponding control pairfed animals had 39% incidence of tumours. These data represent the values obtained 32 weeks after the last administered dose of the carcinogen and indicate the role of vitamin A, both in the initiation as well as in the postinitiation phases of lung carcinogenesis.

Summary The present investigation shows the effect of vitamin A deficiency on the initiation and postinitiation phases of benzo(a)pyrene-induced lung carcinogenesis in male Wistar rats. Lung tumours were induced by giving three intratracheal instillations, one week apart, of 10mg benzo(a)pyrene per instillation. Maximum tumour incidence (100%) and tumour burden per rat was found in rats which were kept on vitamin A deficient diet for 4 weeks prior to the first administration and 8 weeks after the last administration of benzo(a)pyrene. Rats in which vitamin A deficiency was terminated after the last administration of the carcinogen had 83% tumour incidence, whereas corresponding control pairfed animals had 39% incidence of tumours. These data represent the values obtained 32 weeks after the last administered dose of the carcinogen and indicate the role of vitamin A, both in the initiation as well as in the postinitiation phases of lung carcinogenesis.
Today vitamin A is well known for its importance in general growth and differentiation of epithelial tissues and its deficiency has been shown to lead to metaplastic changes in the epithelia of the respiratory, urogenital and gastrointestinal tracts (Harris et al., 1972;Wolback, 1954). These metaplastic changes appeared by light microscopy to be morphologically similar to the changes found in certain precancerous lesions caused by carcinogen administration (Harris et al., 1972;Hayes, 1971). This similarity between histological features of epithelial tissues of vitamin A deficient animals and certain precancerous lesions was the starting point for further investigations in this area. Several investigators have confirmed and extended the early observations and have shown a prophylactic effect of vitamin A on the development of carcinogeninduced epithelial tumours (Bollag & Matter, 1981;Lotan, 1980;Nettesheim & Williams, 1976). However, supplementation with retinoids as a prophylactic agent has resulted in variable effects and these are being related to the problem of distribution and toxicity of vitamin A (Sporn, 1976).  investigated the influence of retinyl acetate supplementation at high dose on the initiation and postinitiation phase of 3-methylcholanthrene induced preneoplastic lung nodules in rats. In this study the incidence of metaplastic lung nodules was found to be 3% in the combined high retinyl acetate dose groups as compared with 42% in the low retinyl acetate dose group, and it was concluded that retinyl acetate treatment had a significant inhibitory effect on the postinitiation phase of preneoplastic lung nodules in rats. However, the effect of vitamin A deficiency on the initiation and postinitiation phases of carcinogenesis has not been evaluated. Therefore, the primary objective of the present investigation was to study and compare the effect of benzo(a)pyrene, a potent environmental lung carcinogen, on tumour incidence in vitamin A deficient rats, both at the initiation and postinitiation phases of pulmonary carcinogenesis.

Materials and methods
Male weanling rats (45-60 g) of our Institute's colony (Wistar derived) were used. They were maintained for 4 weeks on vitamin A deficient casein based diet composed of vitamin-free casein (20%), corn starch (39.8%), dextrose (30%), refined peanut oil (5%), salt mix (4%), choline chloride (0.2%), and vitamin A-free vitamin mix (1.0%). An identical diet supplemented with retinyl acetate (20,000 IU kg-1) was given to control animals. Animals had free access to water and food during the first 4 weeks, thereafter during the next 2 weeks the amount of diet supplied to the control rats was reduced in proportion to that consumed by the deficient group. Equal amounts of benzo(a)pyrene (BP) and Fe203 were mixed in a mortar and ground together (j The Macmillan Press Ltd., 1985 for 2 -4 h, yielding a fine, homogeneously distributed mixture of BP and Fe2O3 (1:1) by weight. This mixture was prepared freshly every week before instillation and was then placed in a 25 ml flat-bottomed conical flask to which 0.15 M NaCl (sterile) was added to make the final concentration of the mixture 50 mg ml -1. After maintaining rats on their respective diets for 4 weeks, a total of three intratracheal instillations were given to each rat with a gap of one week between each administration. Each instillation consisted of 20mg mixture of BP-Fe203/0.4 ml/rat. Animals were instilled under light ether anaesthesia. One day after the third instillation, rats from the vitamin A deficient group were randomly divided into two groups i.e. groups 2 and 3, to receive diet either containing 20,000 IU kg-1 or 1000 IU kg-1 of retinyl acetate respectively. Rats from group 3 were maintained on vitamin A deficient diet (1000IUkg-1) for a further 8 weeks after the last intratracheal instillation of the carcinogen and then were shifted to normal diet containing 20,000 IU retinyl acetate kg-1 diet. In addition to BP-Fe203 treated rats, weight matched rats from normal and vitamin A deficient groups were given 30 mg of Fe203 intratracheally (10mg per instillation, instillations one week apart) to serve as controls. Rats were weighed and food consumption was determined twice a week.
Animals were checked twice daily. Some rats from each group died during study duration and were found cannibalized or exhibited tissue autolysis. These were not evaluated in the study. Twenty or more rats from each group were sacrificed at different time intervals (24, 28 and 32 weeks) after the last instillation of carcinogen. In each case, lungs were removed along with the trachea. Squamous nodules in lungs, >0.5cm or <0.5cm were scored as end points in the study. Lungs were fixed in 10% neutral buffered formalin, processed for routine histological staining with hematoxylin and eosin and examined microscopically.
Liver vitamin A was estimated by the method of Dugan et al. (1964). Statistical analysis of differences was done by Student's t test.

Results
Table I presents the body weight and hepatic vitamin A content of control and vitamin A deficient groups at various time intervals in the study. Initially, all groups were weight matched at 49 +6g per rat. Weight gain in group 3 was slightly less than that in the other groups, but with the overlap of standard deviation between group 3 and other groups, this difference was not significant. Hepatic vitamin A content in rats maintained on vitamin A deficient diet for 4 weeks was reduced to less than 2ugg -1 and was found to be 5pgg-1 in the group of rats (Group 3) which was maintained on a low vitamin A containing diet for a further 8 weeks after the final intratracheal instillation of BP. However, hepatic vitamin A content in all the groups sacrificed on 24, 28, and 32 weeks after the last instillation of the carcinogen, did not show any significant difference. Table II shows tumour incidences in the different groups, sacrificed at weeks 24, 28 and 32 after the Groups 1 through 3 received three intratracheal instillations, one week apart, of 10mg BP at 4 weeks after rats were maintained on diets either containing 20,000 IU kg-1 of retinyl acetate (group 1) or on vitamin A deficient diet (groups 2 and 3). At 6 weeks group 2 was shifted to diet containing 20,000IU of retinyl acetate kg-1, whereas group 3 was fed diet containing 1000IU of retinyl acetate kg-1 till 14 weeks and then this group was also shifted to the diet given to groups 1 and 2. Groups 4 and 5 were maintained on diets given to groups 1 and 3 respectively and received Fe203 instead of BP+Fe203 so as to serve as controls. Weeks 30, 34 and 38 mentioned here corresponds to weeks 24, 28 and 32 after the termination of BP+ Fe203 or Fe203 treatment.
*Values in parentheses are the number of animals in each group at first intratracheal instillation of BP+Fe203 or Fe203.
last BP-Fe203 intratracheal instillation. Tumours were verified histopathologically to be mainly +-'' squamous cell carcinomas (Figure 1). LDetall typing of tumours was not carried out. The 011,0 0 ,majority of the lung tumours were roughly o Q, E 3 spherical and symmetrical (Figure 2). The mean 0 0 C tumour diameter and number of tumours per <,, t4nN tumour-bearing animal (TBA) indicate the relative degrees of tumour burden in the various groups. Fe203 treated groups (Groups 4 and 5) in which =. <, F rats were maintained on the same diets as the rats -o Y a Oin groups 1 and 3 respectively, did not reveal any < ,,X ,,, tumours even after 32 weeks. At 24 weeks, rats from group 1 had the lowest tumour incidence and |3 o 0 T/TBA ratio, whereas these parameters were higher > > 0< .0= <,,"°tumour incidence, however the T/TBA ratio in all = E + < the groups remained almost the same as it was at a, 24 weeks. At 32 weeks, group 1 did not show any CO ;O ,further increase in tumour incidence, whereas it increased from 75% to 83% in group 2, and was 100% in group 3. At this point the T/TBA ratio <,, g = e was increased in all groups, thereby revealing an increased tumour burden per rat. The mortality due to intratracheal instillations of BP- to be maximal in animals kept on vitamin A deficient diet.

Discussion
The role of vitamin A deficiency in tumourigenesis has been studied by several investigators (Nettesheim & Williams, 1976;Rogers et al., 1973;Sporn & Newton, 1981). However, the exact mechanism by which vitamin A influences this process has not been well delineated. In our present study, on periodically killing of rats treated with BP, we have observed that the tumour incidence in vitamin A deficient animals was more than double that in normal pairfed rats. This enhanced susceptibility of the lung to BP-induced carcinogenesis in vitamin A deficiency could possibly be related to a number of factors. Firstly, as changes inflicted by vitamin A deficiency have been shown to be similar to precancerous lesions, vitamin A deficiency may therefore, elevate the initiation and progression of tumourigenic process in animals exposed to chemical carcinogens, as has been observed in our study. Secondly, predisposition of the lung to chemical carcinogenesis may be due to an altered pattern of enzymes involved in the metabolism of these carcinogens, both in lung and liver. BP, like many other polycyclic aromatic hydrocarbons, requires metabolic activation by the action of mixed-function oxidases and vitamin A status has been shown to alter the activities of these enzymes (Becking, 1973;Miranda et al., 1979;Siddik et al., 1980). Our earlier studies in this area (Dogra et al., 1982;Dogra et al., 1983) suggested that imbalance between activation and conjugation processes in lung and liver in vitamin A deficiency might result in an increased yield of carcinogenic metabolites and their slow elimination from the body. This might, in turn, produce a favourable environment for the enhanced binding of reactive carcinogenic metabolites with critical cellular macromolecules like DNA. Earlier, we observed enhanced binding of [3H]-BP to lung DNA in vitamin A deficient rats in in vivo and in vitro studies (Dogra et al., 1984). These studies give support to the argument that mixed-function oxidase enzymes play a very crucial and important role in determining organ susceptibility to tumourigenesis.
In the present study vitamin A deficiency has been found to enhance the process of tumourigenesis, both at the initiation and postinitiation phases. However, the effect of vitamin A deficiency was more prominent at the initiation phase as rats from group 2, killed at 32 weeks after the termination of BP instillation had 83% tumour incidence compared to 39% in normal pairfed rats (group 1). Moreover, the tumour burden per rat was also greater in vitamin A deficient rats than in normal pairfed rats. Furthermore, the enhancing effect of vitamin A deficiency on carcinogenesis during the postinitiation phase was evident from the data on group 3 as rats which were kept on vitamin A deficient diet during and after the challenge with BP, not only had a higher percentage tumour incidence but also the tumour burden per rat was even greater. Also, this higher incidence of tumours was achieved earlier than in the other two groups (groups 1 and 2). This could be due to lack of vitamin A as an essential factor for the normal differentiation of cells during the post initiation phase of tumourigenesis. The other responsible factor could be the slow elimination of carcinogen (given as BP-Fe203) from the lung. Therefore, an effect of vitamin A deficiency on the metabolism of the carcinogen and the penetration of the carcinogens into the target cells could not be ruled out. In conclusion this study has revealed that vitamin A deficiency enhances the process of lung carcinogenesis induced by benzo(a)pyrene both at the initiation and postinitiation phases. However, the more pronounced effect was seen at the initiation stage.