Studies on the genotoxicity of beryllium sulphate in vitro and in vivo

Mutation Research, 240 (1990) 217-225 Elsevier

217

MUTGEN 01522

Studies on the genotoxicity of beryllium sulphate in vitro and in vivo J. A s h b y a, M . I s h i d a t e Jr. b, G . D . S t o n e r c, M . A . M o r g a n c, F. R a t p a n d and R.D. Callander a "Imperial Chemical Industries plc, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SKIO 4TJ (Great Britain), h National Institute of Hygiene Sciences, Tokyo 158 (Japan), c Department of Pathology, Medical College of Ohio, Teledo, OH (U.S.A.) and d Polysar Ltd., Sarnia, Ont. (Canada)

(Received 21 June 1989) (Revision received 17 October 1989) (Accepted 23 October 1989)

Keywords: Beryllium sulphate, genotoxicity; Lung carcinogen

Summary There is limited evidence that beryllium is a lung carcinogen to man, and several compounds of beryllium are carcinogenic to the lungs of the rat, rabbit and monkey. One such compound is beryllium sulphate (BeSO4.4H20). This soluble salt has been evaluated in a range of genotoxicity tests. It was non-mutagenic to Salmonella typhimurium (strains TA1535, 1537, 1538, 98 and 100) when evaluated in the plate-incorporation assay at dose levels up to 5 rag/plate (_+ induced rat-liver $9 mix). It was also non-clastogenic to Chinese hamster lung (CHL) cells cultured in vitro. When dosed to male CBA mice via oral gavage at dose levels of 80% and 50% of the medium lethal dose (2.3 and 1.4 g/kg, respectively) it failed to increase the incidence of micronucleated polychromatic erythrocytes in the bone marrow (sampled at 24, 48 and 72 h post-dosing). However, a marked depression of erythropoiesis was evident 24 h after dosing suggestive of beryllium-mediated bone-marrow toxicity. When tested in the strain A mouse lung tumour bioassay, BeSO 4 induced a significant increase in the number of tumour-bearing animals but not in the number of lung tumours per animal. These findings are discussed within the contexts of other genotoxicity data published for BeSO 4, and of current strategies for the detection of possible human carcinogens.

Beryllium metal and a range of beryllium compounds are carcinogenic to the rat, rabbit and monkey lung. There is also limited evidence of an increased risk of lung cancer in man. The above information led IARC to conclude that beryllium should be considered suspect of being carcino-

Correspondence: Dr. J. Ashby, Imperial Chemical Industries plc, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ (Great Britain).

genic to humans (IARC, 1980). Most experimental data exist for beryllium sulphate (BeSO 4 • 4H20, referred to as BeSO4 hereafter), so this was selected for study. Several authors have suggested that primary attention should be paid to the detection of genotoxic rodent carcinogens as those probably hold the greatest potential for carcinogenicity in man (Kuroki and Matsushima, 1987, Ashby and Tennant, 1988; Bartsch and Malaveille, 1989). Further, Shelby (1988) has reported that the majority

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218

of those agents classified by I A R C as Class I human carcinogens are detected as mutagens by either or both of the Salmonella mutation assay and an assay for rodent bone-marrow clastogenesis, such as the mouse micronucleus assay. It therefore became of interest to evaluate BeSO 4 in these assays. Ldonard and Lauwerys (1987) have reviewed the mutagenicity of BeSO4 in vitro, but no in vivo mutagenicity data have yet been described. The earlier (negative) Salmonella mutagenicity data for BeSO 4 were limited to studies employing low dose levels, consequently it was decided to re-evaluate its mutagenicity to Salmonella as well as studying its activity in a mouse bone-marrow micronucleus assay. In addition, the clastogenicity of this agent to cultured C H L cells (Ishidate et al., 1984) was evaluated, as was its ability to induce lung tumours in strain A mice (Stoner et al., 1985). Materials and methods

Chemicals' Beryllium sulphate ( B e S O 4 . 4 H 2 0 ) was obtained from B D H Ltd., Poole, Dorset (99%) or Wako Pure Chemical Industries Ltd. (99.9%; used for C H L assays), and used as received. Cyclophosphamide (CP) was as described earlier (Ashby and Beije, 1985). Positive control chemicals for the Salmonella assays were exactly as described earlier (Ashby et al., 1985). Salmonella assays Standard plate-incorporation mutation experiments were conducted using strains TA1535, 1537, 1538, 98 and 100 of Salmonella typhimurium as previously described (Ashby et al., 1985). Experiments were conducted in the presence and absence of Aroclor-induced rat liver $9 mix (10%), and each was repeated at least once. Cytogenetic assays Assays were conducted using Chinese hamster lung (CHL) cells in the presence and absence of aroclor induced rat-liver $9 mix as previously described (Ishidate and Odashima, 1977; Ishidate et al., 1984). BeSO4 • 4 H 2 0 was dissolved in physiological saline (0.15 M NaC1). Two exposure periods were employed ( - $ 9 ) and a single exposure

of 6 h with $9 mix, followed by 12 h in its absence; the latter protocol was also conducted in the absence of $9. 100 metaphases were analysed for chromatid and chromosomal aberrations per dose group.

Micronucleus assays Male CBA mice were maintained and treated as described earlier (Ashby and Mirkova, 1987). The test chemical was administered via oral gavage in physiological saline and bone marrow smears prepared and assessed for the incidence of micronucleated polychromatic erythrocytes (MPE) among PE, 24 h, 48 h and 72 h after dosing, as described in detail earlier (Ashby and Mirkova, 1987). 2000 PE were assessed for MPE, per animal, and the ratio of P E / N E was determined based on assessment of 1000 erythrocytes. The dose levels employed were based on the 4-day median lethal dose level (MLD), itself established in preliminary experiments, thus: dose levels of 150, 500 and 1000 m g / k g (oral gavage in physiological saline) yielded 0 / 5 deaths at 4 days, in each case. These dose levels had been indicated by parts of the earlier literature, albeit it is always very difficult to extrapolate such data. In a third toxicity experiment, groups of 5 mice were exposed to a single dose of either 3 g / k g or 5 g / k g of BeSO4. After 4 days, 3 / 5 and 5 / 5 deaths were observed, respectively. Probit analysis of those data indicated 50% and 80% of the M L D to be 1.4 and 2.3 g / k g , respectively (cf. protocol criteria of Heddle et al., 1983). Those dose levels were employed for the first mouse micronucleus assays (Table 3), and no deaths were observed. However, in the second experiment (72 h; Table 3) all of the animals exposed to 80% of the 4 d M L D died, as did 1 / 5 of those exposed to 50% of this dose. Consequently, only the 50% M L D data (1.4 g / k g ) are shown in Table 3. This change in toxicity is unusual, but equally, a wide range of LDs0 values are recorded for derivatives of beryllium (IARC, 1980), and the present M L D in CBA mice is dramatically different from the M T D determined in A / J mice (see below). Lung adenoma A / J strain obtained from MA (U.S.A.).

bioassay male mice, 5 - 6 weeks old, were Jackson Laboratories, Bar Harbor, During the study, the mice were

219 housed in solid bottom and side polycarbonate cages (height 13 cm, width 18 cm and length 28 cm) covered with stainless steel lids and non-woven polyester fibre filter tops. There were 4 animals housed per cage, in accordance with N C I weightspace specifications. Animals were transferred to clean caging twice weekly. Bedding of ground corn cobs was used and the soiled bedding was incinerated. An adequate supply of food (Wayne Lab Blox) was offered ad libitum. Fresh water was supplied ad libitum by twice weekly changes of sanitized water bottles fitted with rubber stoppers and stainless steel sipper tubes. The temperature in the room was maintained at 22 ° + 2 ° C, with a relative humidity of 30-60%. An automatic timing device provided an alternating 12-h cycle of light and darkness. The test chemical was administered by i.p. injection of a solution in distilled water (0.1 m l / m o u s e ) to give the required dose. A maximum tolerated dose (MTD) for BeSO4 was determined. Serial 2-fold dilutions were administered i.p. to groups of 4 mice. The M T D was the maximum dose per injection that all 4 mice survived after receiving 3 i.p. treatments per week for 2 weeks. Animals were held for 1 month before experimental groups were initiated in order to detect delayed toxicity. The M T D determined was 0.1 m g / m o u s e / i n j e c t i o n ( - 5 m g / k g ) . In the tumour bioassay BeSO 4 was tested at 3 doses: the MTD, 0.5 M T D and 0.2 MTD. A vehicle (distilled water) control group was maintained along with the treated groups. Injections were administered 3 times a week for 8 weeks, with 20 mice per group. A small number of animals, 1 from the MTD, 2 from the 0.5 M T D and 2 from the 0.2 MTD, were sacrificed at 26 weeks after initiation of the bioassay, to check for tumours. All remaining animals were sacrificed at 32 weeks after initiation of the bioassay. All data was calculated only on those animals surviving the full 32 weeks. Animals were killed by CO 2 asphyxiation. The lungs were removed and fixed in Tellyesniczky's fluid. The livers were removed, weighed and fixed in 10% buffered formalin. Lung tumours, which appeared as pearly white nodules on the surface of the lung (Shimkin and Stoner, ] 975), were counted using a dissecting microscope and randomly sampled for histopathological evaluation and confirmation of adenoma.

Livers were also examined under a dissecting microscope for the presence of tumours. In addition, the kidneys, spleen, intestines and stomach were examined grossly for any other lesions. The lung-tumour incidence in chemically treated and control animals was compared by Chi square analysis and the turnout multiplicity was compared by one way analysis of variance (ANOVA). Results

Salmonella, CHL and micronucleus assays BeSO 4 was non-mutagenic to the standard tester strains of Salmonella employed (Table 1). The results shown for strains TA1535, 1537 and 100 are representative of repeat experiments. In the first experiment using strain TA1538 a weak effect was seen in the presence, but not in the absence of $9 mix. The corresponding repeat negative data seen for strain TA98 ( + $9) are therefore shown, as are two further experiments using strain TA1538 ( + $9), conducted over a narrower dose range, and which failed to confirm the initial observation. Consequently, BeSO 4 is regarded as non-mutagenic to Salmonella under the present conditions of test. Negative results were observed in the C H L cytogenetic assays (Table 2). It also failed to induce MPE in the bone marrow of mice, despite the production of a depression in erythropoiesis 24 h after dosing. Cyclophosphamide gave the expected positive response 24 h after dosing and the saline controls gave MPE incidences in the normal range (Table 3). A review of previous literature reports on the mutagenicity of BeSO 4 to Salmonella is provided in Table 4. Lung adenoma bioassav Table 5 presents results on the occurrence of lung adenomas in vehicle and treated male A / J mice. Results obtained with the vehicle controls are in close agreement with earlier studies (Shimkin and Stoner, 1975; Stoner and Shimkin, 1982, 1985), and indicate that the occurrence of lung turnouts was not affected by the injections of the vehicle (water). There was a high mortality at the previously determined M T D of BeSO4; 11 mice died before the final sacrifice date. When compared to the vehicle controls, BeSO 4 produced a significant increase in the incidence of

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222 TABLE 3 MALE CBA MOUSE BONE-MARROW M I C R O N U C L E U S ASSAY DATA F O R BeSO 4. H 2 0 Expt. No.

Dose (mg/kg)

Sampling time (h)

Number of animals

P E / N E ( ± SD)

1

Saline (10 m l / k g ) CP (65 m g / k g ) BeSO 4 (1.45 g / k g ) BeSO 4 (2.3 g / k g )

24

5

1.5 ± 0.8

24

5

0.9±0.4

24

5

1.5 + 0.9

1.1 ± 0.4

24

5

0.3 _+0.2 * *

2.0 ± 0.9

Saline (10 ml/kg) BeSO 4 (1.45 g/kg) BeSO4 (2.3 g/kg)

48

5

1.4 + 0.5

1 .l ± 0.5

48

5

1.3 ± 0.2

1.3 ± 0.6

48

5

0.9 _+0.6

1.2 ± 0.8

72

5

1.3 ± 0.7

1.4 ± 0.4

72 (dosed at 48 h) 72

5

0.4±0.1"*

4 a

1.1 ±0.5

2

Saline (10 m l / k g ) CP (65 m g / k g BeSO4 (1.45 g/kg)

MPE/1000 PE ( ± SD) based on 2000 PE assessed per animal 1.4 ± 0.7 12.8±l.2 **

22

±1.7"*

1.1 ±0.2

The dose levels employed represented 50% and 80% of the 4-day MLD. a One animal died at 3 days. Statistical analysis was conducted using a 1-sided Student's ttest; *P < 0.05, * *P < 0.01.

TABLE 4 SUMMARY OF PUBLISHED LITERATURE ON T H E M U T A G E N I C I T Y OF BeSO4 TO STRAINS OF SALMONELLA Investigator

Tester strains of Salmonella typhimurium used (LT 2 and the TA strains listed) LT 2

1530

1535

1536

1537

1538

Comments 98

100

Simmon (1979)

No data presented. Top dose level evaluated 250 #g/plate.

Rosenkranz and Poirier (1979)

No data presented. Only two dose levels evaluated, 25 and 250 ~tg/plate

Simmon et al. (1979)

Host-mediated assay. BeSO 4 administered orally, bacteria by i.p. injection

Tso and Fung (1981)

BeSO4 not tested, rather Be(NO3)z.4H~O, the only other derivative of Be to be assayed in Salmonella. Negative data relate to a spot test. No data shown but the dose range 10-4_10 1 M evaluated, Indications that the top dose level was toxic to wild-type cells.

Present data

Tested to 5000 ~tg/plate, non-mutagenic

All in vitro experiments involved use of Aroclor-induced rat-liver $9 mix. No entry in a column implies no experiments in that strain.

223 TABLE 5 L U N G - T U M O U R RESPONSE IN VEHICLE A N D BeSO4-TREATED A / J M I C E Treatment

Total dose ~ (mg/mouse)

Survivors/ initial

Mice with lung tumours (%)

Average number of lung t u m o u r s / m o u s e

Beryllium sulfate Beryllium sulfate Beryllium sulfate Vehicle (water)

2.40 1.20 0.48 -

8/19 18/18 18/18 20/20

38 c 33 c 17 15

0.38 _+0.48 0.39 _+0.59 0.17 + 0.37 0.15 _+0.36 b

a All mice received 24 i.p. injections of either the vehicle (water) or beryllium sulfate. They were killed 32 weeks after the first injection. h Values are mean_+ standard deviation. Significant increase in the incidence of mice with tumours (Chi squared; R = 5,9 and 4.6 for the middle and high dose levels, respectively).

tumour bearing animals in the two groups receiving total doses of 1.20 (R = 4.6) and 2.40 (R = 5.9) m g / m o u s e . In addition, BeSO 4 produced an increase in the number of lung tumours per mouse in these two groups (i.e. 1.20 and 2.40 m g / m o u s e ) ; however, these increases were not significant ( F = 1.21). There were no liver tumours observed in any of the test or control groups. However, there was evidence of liver toxicity in the high and middle doses of the BeSO4-treated groups. As the dose of BeSO 4 was increased, liver weights were reduced, from 1.33 g per liver in the low dose group, to 1.10 g per liver in the high dose group. In addition to being smaller, the livers were also pale, the lobes showed evidence of adhesions and had rounded edges. All animals treated with the high dose of BeSO 4 showed adhesions throughout the abdomen and ascites. 10 of the animals from the middle dose group (1.2 r a g / m o u s e ) showed some or all of these same characteristics. Livers from the low dose and the vehicle control group appeared normal when examined grossly. Discussion

Beryllium and several of its compounds are toxic to man and carcinogenic to animals (reviews IARC, 1980; L6onard and Lauwerys, 1987). Based on the results of several epidemiological studies, I A R C (1980) have concluded that limited evidence exists for the carcinogenicity of beryllium to the human lung. The site of carcinogenesis of derivatives of beryllium in animals is dependent upon

the route of exposure selected, thus, lung tumours are induced in rats, rabbits and monkeys following either inhalation or intratracheal instillation, and osteosarcomas following either intravenous injection or intraosseus administration (IARC, 1980). A m o n g compounds of beryllium, BeSO 4 has been the best evaluated for genotoxicity and carcinogenicity ( I A R C , 1980; L~onard and Lauwerys, 1987). The present data (Table 1) define BeSO 4 as non-mutagenic to Salmonella when evaluated using standard tester strains in the plate-incorporation assay. These data supplement the results of earlier and limited negative studies (reviewed in Table 4). L6onard and Lauwerys (1987) have suggested that the recent results observed by Marzin and Phi (1985) indicate that the correct strains of Salmonella may not have been used to evaluate the mutagenicity of beryllium and its derivatives. Marzin and Phi (1985) had employed strain TA102 to evaluate 16 derivatives of 10 metals (not including Be), but they only recorded activity for hexavalent chromium salts, and these are active in the standard base-pair mutant strains (TA1535 and TA100; Bianchi et al., 1983). This does not therefore seem to be a serious omission from the database; further, this strain has not been generally adopted due to problems encountered in its use. L~onard and Lauwerys (1987) have conducted a review of the activity of BeSO 4 in mammlian cell in vitro assays; it is active in some assays, but not in all of them. The most extensive data are those of Larramendy et al. (1981) who concluded BeSO 4 to be clastogenic and capable of inducing SCE in

224

both purified human lymphocytes and Syrian hamster cells (at dose levels which also transform the hamster cells). In an earlier study, Paton and Allison (1972) defined BeSO 4 as non-clastogenic to human lymphocytes cultered from whole blood, and Anderson (1983) has subsequently reported a negative SCE response in human lymphocytes, again when using whole blood. Thus, the use of isolated (purified) lymphocytes may be important. Within that context, Larramendy et al. (1981) observed a stronger response for sodium arsenite in purified lymphocytes, as compared to whole blood cells. In contrast, Ray and Altenburg (1978) have reported sodium selenite to be an SCE-inducing agent in whole blood cultures, but not in purified lymphocyte cultures. No evidence of a clastogenic response for BeSO4 was observed in cultured CHL cells in the present experiments. Thus, if BeSO 4 is clastogenic in vitro the conditions of test appear to be critical to the observation of activity. Under routine conditions of test it appears that BeSO4 would probably be recorded as non-clastogenic. The inactivity of BeSO4 in the mouse micronucleus assay was accompanied by a marked depression in bone-marrow erythropoiesis - - itself suggestive of a toxic response of beryllium to the marrow (Table 3). Two metallic human carcinogens, hexavalent chromium and arsenic (as sodium arsenite) are reported to be active in this assay (Wild, 1978; Deknudt et al., 1986; respectively). Given the inactivity of BeSO4 in 3 standard genotoxicity assays, we decided to evaluate it specifically in the strain A mouse lung adenoma bioassay (Shimkin and Stoner, 1975). This assay is sensitive to many carcinogens including certain polycyclic aromatic hydrocarbons, nitrogen and aniline mustards, aziridines, carbamates, nitrosamines and nitrosoureas, etc. (see reviews Stoner and Shimkin, 1982, 1985), and has been recommended as a limited bioassay by Weisburger and Williams (1981). However, in a recent study (Maronpot et al., 1986), the lung-tumour bioassay was found to be insensitive to several chemicals found to be active in long-term rodent carcinogenicity bioassays. Most of these chemicals are liver carcinogens, and the lung-tumour bioassay had been shown earlier to be relatively insensitive to such carcinogens (Shimkin and Stoner, 1975).

BeSO4 produced a significant increase in the lung turnout incidence at total doses of 1.20 and 2.40 mg/mouse. The tumour multiplicity in these groups also increased, however, these increases were not statistically significant. It is possible that BeSO 4 could have induced a significant increase in the tumour multiplicity if the animals had been maintained for longer periods of time, or if higher doses of the compound could have been administered. In addition, the routine use of the i.p. injection route with this bioassay may have compromised the tumour response since lung tumours have only been recorded for BeSO4 following its inhalation (cf. earlier and also, Reeves and Vormald, 1967). It is concluded that BeSO4 is negative in 3 standard genotoxicity assays, and weakly positive for lung-tumour induction in strain A mice. It is suggested that BeSO4 be further evaluated for lung-tumour induction in strain A mice when administered by intratracheal injection or inhalation. In addition, the possible subtle genotoxicity or tumour-promoting activity of BeSO4 to rodent lung cells following its inhalation could be pursued using one of the developing molecular biological techniques (Anderson and Reynolds, 1989). There will always be exceptions to any carcinogen-screening strategy, but these could provide a means of understanding in greater detail the processes of chemically-induced carcinogenesis. Whatever, it appears that BeSO 4 would not be detected as a possible human carcinogen during routine genotoxicity testing.

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