Genetic Toxicology ELSEVIER Mutation Research 368 ( 1996) I2 I - I3 I Biochemical changes associated with the adaptive response of human keratinocytes to N-methyl-N’-nitro-IV-nitrosoguanidine Hanna E. Kleczkowska ‘, Felix R. Althaus * Recelbed 21 September 199.5: revised 2 January 1996: accepted 2 January lY96 Abstract Exposure of cells to low doses of radiation or chemicals renders them more resistant to higher doses of these agents. This phenomenon, termed crdu~~ire r-esporzsr. was studied in quiescent human keratinocytes exposed to the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). The cells were adapted with 2.5 nM MNNG for 60 min and challenged immediately thereafter with 2.5 PM MNNG for 30, 45 or 60 min. Clonogenic survival studies revealed that adapted cells were more resistant to the subsequent challenge treatment (up to 30% higher survival) than unadapted cells. In addition, formation of DNA strand breaks was lower in adapted cells. We monitored poly-ADP-ribosylation activity during expression of the adaptive response both at the substrate as well as the product level. NAD+ utilization in adapted and non-adapted cells exposed to the high dose of MNNG was similar, but recovery from NAD+ depletion was faster in low-dose pretreated cells. Induction of poly(ADP-ribose) formation was more than 2 times higher in low-dose adapted cells and this was associated with the formation of a distinct class of ADP-ribose polymers, i.e.. branched polymers. These polymers exhibit a very high binding affinity for histones and can displace them from DNA. Elevated levels of poly(kDP-ribosel and, particularly, synthesis of branched polymers may play a critical role in low-dose adaptation. K~wwrcl.rt Adaptive response: DNA-alkylating agent: Poly(ADP-ribohyl)ation: Branched polymer 1. Introduction Cells and organisms pre-exposed to low doses of ionizing radiation or chemicals are less susceptible to the damaging effects of higher doses of the same or other agents. This reaction. termed adapri~~ re- .s/7onse, is detectable in higher organisms as a reduc- tion of induced chromosomal aberrations, mutation frequencies, micronuclei formation, DNA strand breaks and/or increased survival (for reviews see Wolff et al.. i 990; Wolff, 1992; Joiner. 1994; Shadley, 1994; Wojcik and Streffer. 1994). Both the duration and chemical concentration of the adapting pretreatment are critical determinants for obtaining a pronounced adaptive response (Sam- . Corresponding author. Tel.: 0011 I 365 I.3 70: Fax: 0041 I 313 01 27. ’ On leave from the Institute of Nuclear Chemistry and Tech- nology, Dorodna 16, PL-03 I95 Warsaw, Poland son and Schwartz, 1980; Morimoto et al., 1986; Shadley and Wiencke, 1989). It has been previously reported that time limits exist between the initial adapting dose and the subsequent challenge dose in 0165.1218/96/$15.00 Q 1996 Elsevier Science B.V. All rights reserved P/f SOl65-1218(96)00003-I