Anomalous genotoxic responses induced in mouse lymphoma L5178Y cells by potassium bromate.
Potassium bromate (KBrO3) is a well-established rodent kidney carcinogen and its oxidising activity is considered to be a significant factor in its mechanism of action. Although it has also been shown to be clearly genotoxic in a range of in vivo and in vitro test systems, surprisingly, it is not readily detected in several cell lines using the standard alkaline Comet assay. However, previous results from this laboratory demonstrated huge increases in tail intensity by modifying the method to include incubation with either human 8-oxodeoxyguanosine DNA glycosylase-1 (hOGG1) or bacterial formamidopyrimidine DNA glycosylase (FPG) indicating that, as expected, significant amounts of 8-oxodeoxyguanosine (8-OHdG) were induced. The purpose of this work, therefore, was to investigate why KBrO3, in contrast to other oxidising agents, gives a relatively poor response in the standard Comet assay. Results confirmed that it is a potent genotoxin in mouse lymphoma L5178Y cells inducing micronuclei and mutation at the tk and hprt loci at relatively non-cytotoxic concentrations. Subsequent time-course studies demonstrated that substantial amounts of 8-OHdG appear to remain in cells 24h after treatment with KBrO3 but result in no increase in frank stand breaks (FSB) even though phosphorylated histone H2AX (gamma-H2AX) antibody labelling confirmed the presence of double-strand breaks. Using bromodeoxyuracil (BrdU) incorporation together with measured increases in cell numbers, L5178Y cells also appeared to go through the cell cycle with unrepaired hOGG1-recognisable damage. Since unrepaired 8-OHdG can give rise to point mutations through G:C-->T:A transversions, it was also surprising that mutation could not be detected at the Na+/K+ATPase locus as determined by ouabain resistance. Some increases in strand breakage could be seen in the Comet assay by increasing the unwinding time, but only at highly toxic concentrations and to a much smaller extent than would be expected from the magnitude of the other genotoxic responses. It was considered unlikely that these anomalous observations were due to the inability of L5178Y cells to recognise 8-OHdG because these cells were shown to express mOGG1 and have functional cleavage activity at the adducted site. It appears that the responses of L5178Y cells to KBrO3 are complex and differ from those induced by other oxidising agents.
Safety Assessment, AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK104TG, UK. catherine.priestley@astrazeneca.com
Catherine C
CC
Priestley
Richard M
RM
Green
Michael D
MD
Fellows
Ann T
AT
Doherty
Nikolas J
NJ
Hodges
Michael R
MR
O'Donovan
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D001959:Bromates / Q000633:toxicity*; D045744:Cell Line, Tumor; D020552:Comet Assay / Q000379:methods; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008223:Lymphoma; D051379:Mice; D015162:Micronucleus Tests; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001959:Bromates; D003849:Deoxyguanosine; D006657:Histones; D009153:Mutagens; C019536:potassium bromate; C475961:gamma-H2AX protein, mouse