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The use of cultured mammalian cells in the UKEMS collaborative genotoxicity trial (1981)
Mutation Research, 100 (1982) 271-276
271
Elsevier Biomedical Press
T H E USE OF C U L T U R E D M A M M A L I A N CELLS IN T H E UKEMS C O L L A B O R A T I V E G E N O T O X I C I T Y TRIAL (1981)
C.F. ARLETT (Co-ordinator)
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BNI 9QG, East Sussex (Great Britain) (Received 9 October 1981) (Accepted 13 October 1981)
In the light of the considerable costs involved in performing even the simplest experiments with mammalian cells in culture we are fortunate in having such a considerable body of data available for study. Details of the experimental methods, protocols, full results and the participants own interpretation of their results are given in the individual reports. The mammalian cell tests fell into 3 groups; the mutation tests, 7 laboratories participated, cell transformation, 4 laboratories participated and damage to DNA and its repair, here 3 laboratories provided data. It seems appropriate to present the summarised data under these separate headings. MUTATION TESTS
A list of contributors, systems used and chemicals studied is provided in Table 1. A gross evaluation as a positive or negative result is also included. 2 species, the mouse and Chinese hamster provided cells for use in these assays. From the mouse, 2 lymphoma cell lines, P388 and L5178Y were used, one laboratory used P388 and two used L5178Y. It should be recognised that while designated L5178Y the cells and the ways in which they are used bear little relation to each other. With the Chinese hamster, 3 laboratories used V79 (male lung cells) and again it should be noted that the cells were obtained from a variety of different sources by each laboratory and since they may change under local culture condition the cells should not be considered identical. One study employed CHO (ovary) cells. With P388, Anderson and Cross (1982) investigated 4CMB as an inducer of 5-iodo-2'-deoxyuridine (IUdR) resistance in the presence or absence of $9 mix, 2 Expts. were performed. 4CMB induced resistance to IUdR. For L5178Y, Ross and McGregor (1982) produced data for the induction of TFT resistance in 3 Expts. performed with 4CMB, 4HMB and BC in the presence or absence of $9 mix. 4CMB alone produced a dose-response curve in the absence of $9 mix. Arlett et al. (1982) investigated the induction of TG, OUA, ara-C and MTX resistance in L5178Y cells by 4CMB in the absence of $9 mix. A number of experiments were performed and 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
272 TABLE 1 MAMMALIAN MUTATION TESTS, A SUMMARY CHEMICALS TESTED AND RESULTS Authors and affiliation
$9 mix
4CMB
Anderson and Cross, ICI Central Toxicology
+ -
Ross and McGregor, Inveresk Research
+ .
A r l e t t e t a l . , M R C Cell Mutation Unit
-
OF
SOURCE
INFORMATION,
BC
Mutation assay
+ ND + ND (reduced)
ND ND
P388 resistance to IUdR
+
-
-
L5178Y resistance to TFT
-
ND ND ND ND
ND ND ND ND
+
ND
ND
V79 resistance to 8-AzaG or 6-TG
+
+
--
+
V79 resistance to 8-AzaG
Mirzayans et al., Genetics, Swansea
-
+ +?
-
+ +?
Phillips and James, BIBRA
+
+
.
.
.
+ + -
Fox, Paterson Laboratories
Lee and Webber, Chemical Defence Establishment
4HMB
OF
L5178Yresistanceto araC MTX OUA 6-TG
V79 resistance to 8-AzaG OUA CHO resistance to 6-TG
+, positive; - , negative; + 2, very slight induction; ND, not done.
4CMB was shown to induce MTX- and ara-C- but not OUA-resistant mutants. The data for TG resistance was insufficient for a decision. W i t h V 7 9 cells F o x ( 1 9 8 2 ) p r o v i d e d d a t a f r o m 3 s e p a r a t e e x p e r i m e n t s f o r 4 C M B in the absence
of $9 mix,
selecting
for mutants
with either 8-azaguanine
or
6-thioguanine. A positive response was obtained whichever selective agent was used, t h e r e s u l t s w e r e less c o n v i n c i n g w i t h T G - a r e s u l t w h i c h is c o n s i s t e n t w i t h t h a t o f A r l e t t et al. ( 1 9 8 2 ) w i t h L 5 1 7 8 Y cells. L e e a n d W e b b e r ( 1 9 8 2 ) s e l e c t e d 8 - a z a g u a n i n e r e s i s t a n c e i n V 7 9 cells t r e a t e d w i t h 4 C M B , 4 H M B a n d B C i n t h e p r e s e n c e o f $ 9 m i x , results for 2 Expts. are provided. Both 4CMB and BC induced a dose-dependent increase in the recovery of mutants, 4HMB was not mutagenic. The third V79 study w a s p r o v i d e d b y M i r z a y a n s et al. (1982) it d i f f e r e d f r o m t h e o t h e r s a n d t h e m o u s e cells i n e m p l o y i n g HGPRT
the in situ method.
Resistance to OUA
and mutation
system were investigated in the absence of $9 mix. Both 4CMB
induced 8-azaguanine-resistant mutants in a dose-dependent
in the and BC
fashion, BC was more
273 effective than 4CMB at equitoxic doses. The effect of 4CMB was remarkably more substantial than in the study reported by Fox (1982) and may represent a major difference resulting from the 2 experimental protocols. The single study with C H O cells (Phillips and James, 1982) reported attempts to induce TG resistance with 4CMB (4 Expts.), 4HMB and BC (2 Expts. each) in the absence of $9 mix. The data appeared somewhat inconsistent but indicated a positive response for 4CMB and BC in a narrow window in the dose scale. Overall, the results with these mammalian mutation tests are consistent in indicating positive mutagenic activity for 4CMB in the presence or absence of $9 mix. Only 4 groups tested 4HMB but all reported negative results. Some, but not all, studies included positive or negative controls as well as the obvious comparisons between test compounds. While accepting 4CMB as a positive mutagen no systematic attempt (except by Arlett et al., 1982) was made to scale its effect. These authors regard 4CMB as being more effective than EMS and less effective than 4NQO at equitoxic doses in the induction of ara-C-resistant mutants. This study is also of interest in showing that 4CMB does not mutate the OUA system, a similar result was tentatively suggested by Mirzayans et al. (1982) and may imply that 4CMB is not acting as a frame-shift mutagen in the mammalian cells (Arlett, 1981). In 3 out of 4 studies BC also proved mutagenic, however, in these, its effectiveness could not be scaled against 4CMB. CELL TRANSFORMATION A list of contributors, systems used and chemicals studied is provided in Table 2 together with a gross evaluation of the results. Using the BHK system in the absence of $9 mix, Styles and Penman (1982) obtained a positive dose-dependent response with 4CMB and a zero response with BC. On the other hand the study of Dehnel et al. (1982) gave equivocal results with 4CMB and a weak positive result for BC in the absence of $9 mix. 4HMB was clearly positive in this investigation. A third study of the BHK system by Poole and McGregor (1982a) of all 3 agents in the presence or absence of $9 mix, showed a significant transformation effect for 4CMB in the absence of $9 and BC in the presence of $9 mix. These particular results are of some importance with respect to an evaluation of the BHK-transformation assay. The inconsistencies were more substantial than observed with mammalian mutation tests despite there being a greater variety of such tests and indicate that the BHK-transformation test 'does not travel well' (Daniel and Dehnel, 1980). The positive result for 4HMB obtained by Dehnel et al. (1982) is to be contrasted with 4 negative results in the mutation tests and 2 negative results from transformation tests. On the other hand BC appeared to be weakly positive for both the mutation and BHK transformation tests. 2 additional transformation systems were also used. Poole and McGregor (1982b) investigated the response of the C 3 H / 1 0 T ½ mouse system with all 3 agents in the
274 TABLE 2 TRANSFORMATION ASSAYS, A SUMMARY OF THE SOURCE OF INFORMATION, CHEMICALS TESTED AND RESULTS Authors and affiliation
$9 mix
4CMB
4HMB
BC
System
Styles and Penman, ICI Central Toxicology
-
+
ND
-
BHK
Dehnel et al., Huntingdon Research Centre
+
no clear response no clear response
+
less
BHK
+
+/-
-
Poole and McGregor, Inveresk Research
+ -
+/-
-
+
BHK
Poole and McGregor, Inveresk Research
+ -
+
-
-
mouse C3H/10TV2
Hatch et al., Northrop Services
-
+
ND
ND
hamster embryo cells viral transformation
+, positive; - , negative; + / - , equivocal result.
presence or absence of $9 mix a n d f o u n d a t r a n s f o r m a t i o n effect only for 4 C M B in the absence of $9 mix. H a t c h et al. (1982) using a viral t r a n s f o r m a t i o n assay in h a m s t e r e m b r y o cells tested 4 C M B in the absence o f $9 mix a n d showed a positive d o s e - d e p e n d e n t e n h a n c e m e n t o f t r a n s f o r m a t i o n . These a d d i t i o n a l studies c o n f i r m the positive response for 4 C M B a n d give less overall weight to the equivocal results o b t a i n e d by Dehnel et al. (1982) for this c o m p o u n d . REPAIR TESTS A s u m m a r y of c o n t r i b u t o r s , systems used a n d chemicals studied is provided in T a b l e 3 with a n e v a l u a t i o n of the results. M i r z a y a n s et al. (1982) provided data o n the i n d u c t i o n a n d repair o f strand breaks in the presence a n d absence o f cytosine a r a b i n o s i d e (araC) by the three test c o m p o u n d s in the absence of S9 mix using h u m a n alveolar cells. A clear d e m o n s t r a t i o n o f s t r a n d break i n d u c t i o n a n d its repair was seen for BC. F o r 4 C M B a class of d a m a g e was i n d u c e d whose repair can be inhibited by araC. P e a r s o n a n d E d w a r d s (1982) provided data for 4 C M B for 3 repair assays in the absence of $9 mix for H e L a $3 cells. These results showed that strand breakage as m e a s u r e d by alkaline elution was increased by 4CMB. There were n o effects o n u n s c h e d u l e d D N A synthesis or repair replication. Barrett (1982) p r o d u c e d data o n u n s c h e d u l e d D N A synthesis in H e L a $3 cells following t r e a t m e n t with the 3 test c o m p o u n d s in the presence or absence of $9 mix. 4 C M B alone gave a positive result.
275
TABLE 3 DNA
DAMAGE
CHEMICALS
AND
ITS R E P A I R ,
A SUMMARY
OF THE
SOURCE
OF INFORMATION,
TESTED AND RESULTS
Authors and affiliation
$9 mix
4HMB
BC
-
-
+
+
0
+
+ -
ND ND
ND ND
(i) strand breakage (ii) u n s c h e d u l e d D N A
-
ND
ND
synthesis (iii) repair replication
Mirzayans et al., Genetics, Swansea
Pearson and Edwards, ICI Central Toxicology
-
human alveolar cells (i) induction of strand breaks in absence of araC (ii) repair of strand breaks in presence of araC h u m a n H e L a $3
Barrett, Boots
+ , positive; - ,
Test
4CMB
+
+
--
+
human H e L a $3 unscheduled D N A synthesis
n e g a t i v e ; N D , not done; 0, no damage therefore no repair.
This effect on unscheduled D N A synthesis must be contrasted with the experiments o f Pearson and Edwards (1982) although the assay systems themselves were quite different. Less information is available from the repair studies but confirm the genotoxic action o f 4CMB and the absence of any action by 4HMB. The results with BC by Mirzayans et al. (1982) and Barrett (1982) are contradictory, however, the authors were looking at different end-points. GENERAL
CONCLUSION
Given that no special weighting may be attributed to any particular set of observations, 17 out of 26 tests were positive for 4CMB. The mutation tests gave the highest ratio of positive results. With 4 H M B 2 tests by Dehnel et al. (1982) on BHK transformations were positive out of a total of 16, suggesting that this c o m p o u n d was indeed not a genotoxic agent. BC gave 8 positive out of a total o f 17 tests, suggesting that more studies with mammalian cells are necessary before a definite conclusion can be reached with this compound.
276 REFERENCES Anderson, D., and M.F. Cross (1982) Studies with 4CMB in P388 cells selected in 5-iodo-2'-deoxyuridine, Mutation Res., 100, 257-261. Arlett, C.F. (1981) Genetic markers for mutagenesis studies in mammalian cells, in: P.C. Hanawalt and E.C. Friedberg (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York, pp. 566-568. Arlett, C.F., W.J. Muriel, J. Cole and J. Lowe (1982) The induction of mutants in L5178Y mouse lymphoma cells by 4-chloromethylbiphenyl, Mutation Res., 100, 253-256. Barrett, R.H. (1982) UKEMS trial compounds: induction of unscheduled DNA synthesis in HeLa $3 cells, Mutation Res., 100, 207-209. Daniel, M.R., and J.M. Dehnel (1980) Factors affecting the performance of the Styles cell transformation test, Carcinogenesis, 1,657-667. Dehnel, J.M., M.R. Daniel and M. Richold (1982) A comparison of the response of BHK21 C13 cells to 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl and benzyl chloride in a cell transformation test, Mutation Res., 100, 223-226. Fox, M. (1982) Cytotoxic and mutagenic effects at the HGPRT locus of 4CMB in V79 Chinese hamster cells, Mutation Res., 100, 235-238. Hatch, G.G., P.D. Mamay and S. Nesnow (1982) Enhancement of viral transformation of hamster embryo cells by pretreatment with 4CMB, Mutation Res., 100, 229-233. Lee, C.G., and T.D. Webber (1982) Effects of benzyl chloride, 4-chloromethylbiphenyl, and 4-hydroxymethylbiphenyl on the mutation of V79 cells to azaguanine resistance, Mutation Res., 100, 245-248. Mirzayans, F., P.J. Davies and J.M. Parry (1982) The cytotoxic and mutagenic effects of 4CMB, BC and 4HMB in V79 Chinese hamster cells, Mutation Res., 100, 239-244. Mirzayans, R., J. Meredith and R. Waters (1982) DNA damage and its repair in cultured human alveolar tumor cells treated with benzyl chloride, 4-chloromethylbiphenyl or 4-hydroxymethylbiphenyl, Mutation Res., 100, 203-206. Pearson, C.M., and K. Edwards (1982) An investigation of DNA-repair phenomena induced by 4-chloromethylbiphenyl (4CMB), Mutation Res., 100, 211-214. Phillips, B.J., and T.E.B. James (1982) The effects of 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyl and benzyl chloride on SCE, chromosome aberration and point mutation in cultured Chinese hamster ovary cells, Mutation Res., 100, 263-269. Poole, A. and D.B. McGregor (1982a) Transformation of BHK 21 C13 cells by BC, 4CMB and 4HMB using the method of Styles, Mutation Res., 100, 215-217. Poole, A., and D.B. McGregor (1982b) Induction of morphological transformation in C3H/10TI/2 clone 8 cells, Mutation Res., 100, 219-221. Ross, C.A., and D.B. McGregor (1982) Mutation at the TK locus of mouse lymphoma L5178Y cells by 4-chloromethylbiphenyl, 4-hydroxymethylbiphenyland benzyl chloride, Mutation Res., 100, 249-25 I. Styles, J., and S. Penman (1982) The activity of 4-chloromethylbiphenyl in the BHK cell transformation assay, Mutation Res., I00, 227-228.
271
Elsevier Biomedical Press
T H E USE OF C U L T U R E D M A M M A L I A N CELLS IN T H E UKEMS C O L L A B O R A T I V E G E N O T O X I C I T Y TRIAL (1981)
C.F. ARLETT (Co-ordinator)
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BNI 9QG, East Sussex (Great Britain) (Received 9 October 1981) (Accepted 13 October 1981)
In the light of the considerable costs involved in performing even the simplest experiments with mammalian cells in culture we are fortunate in having such a considerable body of data available for study. Details of the experimental methods, protocols, full results and the participants own interpretation of their results are given in the individual reports. The mammalian cell tests fell into 3 groups; the mutation tests, 7 laboratories participated, cell transformation, 4 laboratories participated and damage to DNA and its repair, here 3 laboratories provided data. It seems appropriate to present the summarised data under these separate headings. MUTATION TESTS
A list of contributors, systems used and chemicals studied is provided in Table 1. A gross evaluation as a positive or negative result is also included. 2 species, the mouse and Chinese hamster provided cells for use in these assays. From the mouse, 2 lymphoma cell lines, P388 and L5178Y were used, one laboratory used P388 and two used L5178Y. It should be recognised that while designated L5178Y the cells and the ways in which they are used bear little relation to each other. With the Chinese hamster, 3 laboratories used V79 (male lung cells) and again it should be noted that the cells were obtained from a variety of different sources by each laboratory and since they may change under local culture condition the cells should not be considered identical. One study employed CHO (ovary) cells. With P388, Anderson and Cross (1982) investigated 4CMB as an inducer of 5-iodo-2'-deoxyuridine (IUdR) resistance in the presence or absence of $9 mix, 2 Expts. were performed. 4CMB induced resistance to IUdR. For L5178Y, Ross and McGregor (1982) produced data for the induction of TFT resistance in 3 Expts. performed with 4CMB, 4HMB and BC in the presence or absence of $9 mix. 4CMB alone produced a dose-response curve in the absence of $9 mix. Arlett et al. (1982) investigated the induction of TG, OUA, ara-C and MTX resistance in L5178Y cells by 4CMB in the absence of $9 mix. A number of experiments were performed and 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
272 TABLE 1 MAMMALIAN MUTATION TESTS, A SUMMARY CHEMICALS TESTED AND RESULTS Authors and affiliation
$9 mix
4CMB
Anderson and Cross, ICI Central Toxicology
+ -
Ross and McGregor, Inveresk Research
+ .
A r l e t t e t a l . , M R C Cell Mutation Unit
-
OF
SOURCE
INFORMATION,
BC
Mutation assay
+ ND + ND (reduced)
ND ND
P388 resistance to IUdR
+
-
-
L5178Y resistance to TFT
-
ND ND ND ND
ND ND ND ND
+
ND
ND
V79 resistance to 8-AzaG or 6-TG
+
+
--
+
V79 resistance to 8-AzaG
Mirzayans et al., Genetics, Swansea
-
+ +?
-
+ +?
Phillips and James, BIBRA
+
+
.
.
.
+ + -
Fox, Paterson Laboratories
Lee and Webber, Chemical Defence Establishment
4HMB
OF
L5178Yresistanceto araC MTX OUA 6-TG
V79 resistance to 8-AzaG OUA CHO resistance to 6-TG
+, positive; - , negative; + 2, very slight induction; ND, not done.
4CMB was shown to induce MTX- and ara-C- but not OUA-resistant mutants. The data for TG resistance was insufficient for a decision. W i t h V 7 9 cells F o x ( 1 9 8 2 ) p r o v i d e d d a t a f r o m 3 s e p a r a t e e x p e r i m e n t s f o r 4 C M B in the absence
of $9 mix,
selecting
for mutants
with either 8-azaguanine
or
6-thioguanine. A positive response was obtained whichever selective agent was used, t h e r e s u l t s w e r e less c o n v i n c i n g w i t h T G - a r e s u l t w h i c h is c o n s i s t e n t w i t h t h a t o f A r l e t t et al. ( 1 9 8 2 ) w i t h L 5 1 7 8 Y cells. L e e a n d W e b b e r ( 1 9 8 2 ) s e l e c t e d 8 - a z a g u a n i n e r e s i s t a n c e i n V 7 9 cells t r e a t e d w i t h 4 C M B , 4 H M B a n d B C i n t h e p r e s e n c e o f $ 9 m i x , results for 2 Expts. are provided. Both 4CMB and BC induced a dose-dependent increase in the recovery of mutants, 4HMB was not mutagenic. The third V79 study w a s p r o v i d e d b y M i r z a y a n s et al. (1982) it d i f f e r e d f r o m t h e o t h e r s a n d t h e m o u s e cells i n e m p l o y i n g HGPRT
the in situ method.
Resistance to OUA
and mutation
system were investigated in the absence of $9 mix. Both 4CMB
induced 8-azaguanine-resistant mutants in a dose-dependent
in the and BC
fashion, BC was more
273 effective than 4CMB at equitoxic doses. The effect of 4CMB was remarkably more substantial than in the study reported by Fox (1982) and may represent a major difference resulting from the 2 experimental protocols. The single study with C H O cells (Phillips and James, 1982) reported attempts to induce TG resistance with 4CMB (4 Expts.), 4HMB and BC (2 Expts. each) in the absence of $9 mix. The data appeared somewhat inconsistent but indicated a positive response for 4CMB and BC in a narrow window in the dose scale. Overall, the results with these mammalian mutation tests are consistent in indicating positive mutagenic activity for 4CMB in the presence or absence of $9 mix. Only 4 groups tested 4HMB but all reported negative results. Some, but not all, studies included positive or negative controls as well as the obvious comparisons between test compounds. While accepting 4CMB as a positive mutagen no systematic attempt (except by Arlett et al., 1982) was made to scale its effect. These authors regard 4CMB as being more effective than EMS and less effective than 4NQO at equitoxic doses in the induction of ara-C-resistant mutants. This study is also of interest in showing that 4CMB does not mutate the OUA system, a similar result was tentatively suggested by Mirzayans et al. (1982) and may imply that 4CMB is not acting as a frame-shift mutagen in the mammalian cells (Arlett, 1981). In 3 out of 4 studies BC also proved mutagenic, however, in these, its effectiveness could not be scaled against 4CMB. CELL TRANSFORMATION A list of contributors, systems used and chemicals studied is provided in Table 2 together with a gross evaluation of the results. Using the BHK system in the absence of $9 mix, Styles and Penman (1982) obtained a positive dose-dependent response with 4CMB and a zero response with BC. On the other hand the study of Dehnel et al. (1982) gave equivocal results with 4CMB and a weak positive result for BC in the absence of $9 mix. 4HMB was clearly positive in this investigation. A third study of the BHK system by Poole and McGregor (1982a) of all 3 agents in the presence or absence of $9 mix, showed a significant transformation effect for 4CMB in the absence of $9 and BC in the presence of $9 mix. These particular results are of some importance with respect to an evaluation of the BHK-transformation assay. The inconsistencies were more substantial than observed with mammalian mutation tests despite there being a greater variety of such tests and indicate that the BHK-transformation test 'does not travel well' (Daniel and Dehnel, 1980). The positive result for 4HMB obtained by Dehnel et al. (1982) is to be contrasted with 4 negative results in the mutation tests and 2 negative results from transformation tests. On the other hand BC appeared to be weakly positive for both the mutation and BHK transformation tests. 2 additional transformation systems were also used. Poole and McGregor (1982b) investigated the response of the C 3 H / 1 0 T ½ mouse system with all 3 agents in the
274 TABLE 2 TRANSFORMATION ASSAYS, A SUMMARY OF THE SOURCE OF INFORMATION, CHEMICALS TESTED AND RESULTS Authors and affiliation
$9 mix
4CMB
4HMB
BC
System
Styles and Penman, ICI Central Toxicology
-
+
ND
-
BHK
Dehnel et al., Huntingdon Research Centre
+
no clear response no clear response
+
less
BHK
+
+/-
-
Poole and McGregor, Inveresk Research
+ -
+/-
-
+
BHK
Poole and McGregor, Inveresk Research
+ -
+
-
-
mouse C3H/10TV2
Hatch et al., Northrop Services
-
+
ND
ND
hamster embryo cells viral transformation
+, positive; - , negative; + / - , equivocal result.
presence or absence of $9 mix a n d f o u n d a t r a n s f o r m a t i o n effect only for 4 C M B in the absence of $9 mix. H a t c h et al. (1982) using a viral t r a n s f o r m a t i o n assay in h a m s t e r e m b r y o cells tested 4 C M B in the absence o f $9 mix a n d showed a positive d o s e - d e p e n d e n t e n h a n c e m e n t o f t r a n s f o r m a t i o n . These a d d i t i o n a l studies c o n f i r m the positive response for 4 C M B a n d give less overall weight to the equivocal results o b t a i n e d by Dehnel et al. (1982) for this c o m p o u n d . REPAIR TESTS A s u m m a r y of c o n t r i b u t o r s , systems used a n d chemicals studied is provided in T a b l e 3 with a n e v a l u a t i o n of the results. M i r z a y a n s et al. (1982) provided data o n the i n d u c t i o n a n d repair o f strand breaks in the presence a n d absence o f cytosine a r a b i n o s i d e (araC) by the three test c o m p o u n d s in the absence of S9 mix using h u m a n alveolar cells. A clear d e m o n s t r a t i o n o f s t r a n d break i n d u c t i o n a n d its repair was seen for BC. F o r 4 C M B a class of d a m a g e was i n d u c e d whose repair can be inhibited by araC. P e a r s o n a n d E d w a r d s (1982) provided data for 4 C M B for 3 repair assays in the absence of $9 mix for H e L a $3 cells. These results showed that strand breakage as m e a s u r e d by alkaline elution was increased by 4CMB. There were n o effects o n u n s c h e d u l e d D N A synthesis or repair replication. Barrett (1982) p r o d u c e d data o n u n s c h e d u l e d D N A synthesis in H e L a $3 cells following t r e a t m e n t with the 3 test c o m p o u n d s in the presence or absence of $9 mix. 4 C M B alone gave a positive result.
275
TABLE 3 DNA
DAMAGE
CHEMICALS
AND
ITS R E P A I R ,
A SUMMARY
OF THE
SOURCE
OF INFORMATION,
TESTED AND RESULTS
Authors and affiliation
$9 mix
4HMB
BC
-
-
+
+
0
+
+ -
ND ND
ND ND
(i) strand breakage (ii) u n s c h e d u l e d D N A
-
ND
ND
synthesis (iii) repair replication
Mirzayans et al., Genetics, Swansea
Pearson and Edwards, ICI Central Toxicology
-
human alveolar cells (i) induction of strand breaks in absence of araC (ii) repair of strand breaks in presence of araC h u m a n H e L a $3
Barrett, Boots
+ , positive; - ,
Test
4CMB
+
+
--
+
human H e L a $3 unscheduled D N A synthesis
n e g a t i v e ; N D , not done; 0, no damage therefore no repair.
This effect on unscheduled D N A synthesis must be contrasted with the experiments o f Pearson and Edwards (1982) although the assay systems themselves were quite different. Less information is available from the repair studies but confirm the genotoxic action o f 4CMB and the absence of any action by 4HMB. The results with BC by Mirzayans et al. (1982) and Barrett (1982) are contradictory, however, the authors were looking at different end-points. GENERAL
CONCLUSION
Given that no special weighting may be attributed to any particular set of observations, 17 out of 26 tests were positive for 4CMB. The mutation tests gave the highest ratio of positive results. With 4 H M B 2 tests by Dehnel et al. (1982) on BHK transformations were positive out of a total of 16, suggesting that this c o m p o u n d was indeed not a genotoxic agent. BC gave 8 positive out of a total o f 17 tests, suggesting that more studies with mammalian cells are necessary before a definite conclusion can be reached with this compound.
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