Etheno Adducts In DNA Research Articles

The AlkB Family of Fe (II)/Alpha-Ketoglutarate-Dependent Dioxygenases Modulates Embryogenesis through Epigenetic Regulation

The study of epigenetic regulation has made substantial progress in recent years. The AlkB family in E. coli was identified as a type of DNA repair enzyme that removes alkyl adducts from nucleobases. Recently, nine mammalian homologs, ALKBH1-9, have been successfully identified and defined as diverse demethylases. ALKBH1, ALKBH5, ALKBH8 and ALKBH9 act as RNA demethylases, while ALKBH2-3 and ALKBH7 correct methyl and etheno adducts in DNA. Moreover, ALKBH4 focuses primarily on actin. Disorders of AlkB family level in mammals induce many types of diseases. In this review, we will elaborate on the structure and biological function of the members of the AlkB family. We will also focus on the latest progress of the research on the mammalian AlkB family, particularly on new breakthroughs, and present the relevant disorders or diseases induced by an abnormal level of the AlkB family. The AlkB family plays a crucial role in embryogenesis and differentiation. The aberrant level of the AlkB family leads to many types of diseases. The members of the AlkB family may serve as potential cancer markers and possible therapeutic targets in the future.

Non-homologous functions of the AlkB homologs.

The DNA repair enzyme AlkB was identified in E. coli more than three decades ago. Since then, nine mammalian homologs, all members of the superfamily of alpha-ketoglutarate and Fe(II)-dependent dioxygenases, have been identified (designated ALKBH1-8 and FTO). While E. coli AlkB serves as a DNA repair enzyme, only two mammalian homologs have been confirmed to repair DNA in vivo. The other mammalian homologs have remarkably diverse substrate specificities and biological functions. Substrates recognized by the different AlkB homologs comprise erroneous methyl- and etheno adducts in DNA, unique wobble uridine modifications in certain tRNAs, methylated adenines in mRNA, and methylated lysines on proteins. The phenotypes of organisms lacking or overexpressing individual AlkB homologs include obesity, severe sensitivity to inflammation, infertility, growth retardation, and multiple malformations. Here we review the present knowledge of the mammalian AlkB homologs and their implications for human disease and development.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer

Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10wk post-infection, progressing into colon carcinoma by 20wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

The DNA dioxygenase ALKBH2 protects Arabidopsis thaliana against methylation damage

The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The mammalian AlkB homologues ALKBH2 and ALKBH3 display EcAlkB-like repair activity in vitro, but their substrate specificities are different, and ALKBH2 is the main DNA repair enzyme for 1-meA in vivo. The genome of the model plant Arabidopsis thaliana encodes several AlkB homologues, including the yet uncharacterized protein AT2G22260, which displays sequence similarity to both ALKBH2 and ALKBH3. We have here characterized protein AT2G22260, by us denoted ALKBH2, as both our functional studies and bioinformatics analysis suggest it to be an orthologue of mammalian ALKBH2. The Arabidopsis ALKBH2 protein displayed in vitro repair activities towards methyl and etheno adducts in DNA, and was able to complement corresponding repair deficiencies of the E. coli alkB mutant. Interestingly, alkbh2 knock-out plants were sensitive to the methylating agent methylmethanesulphonate (MMS), and seedlings from these plants developed abnormally when grown in the presence of MMS. The present study establishes ALKBH2 as an important enzyme for protecting Arabidopsis against methylation damage in DNA, and suggests its homologues in other plants to have a similar function.

Simultaneous Quantification of Three Lipid Peroxidation-Derived Etheno Adducts in Human DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry

Etheno DNA adducts are promutagenic DNA lesions derived from exogenous industrial chemicals, as well as endogenous sources including lipid peroxidation. Furthermore, levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues. In this study, we have developed a highly sensitive and specific stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) assay for simultaneous detection and accurate quantification of 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo), 3,N(4)-etheno-2'-deoxycytidine (epsilondCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in tissue DNA. Typically, [(13)C(1),(15)N(2)]epsilondAdo, [(15)N(3])epsilondCyd, and [(13)C(1),(15)N(2)]1,N(2)-epsilondGuo were added to calf thymus, human placenta, or human leukocyte DNA as internal standards, and the mixture was subjected to enzyme hydrolysis to form the nucleosides. The etheno adducts in DNA hydrolysate were enriched by a reversed phase solid-phase extraction column before analysis by nanoLC-NSI/MS/MS under the highly selective reaction monitoring (H-SRM) mode. This nanoLC-NSI/MS/MS assay achieved attomole-level sensitivity with the detection limit of 0.73, 160, and 34 amol for the respective standard epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo injected on-column, while the quantification limit for the entire assay was 0.18, 4.0, and 3.4 fmol, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in human placental DNA were 28.2, 44.1, and 8.5 adducts in 10(8) normal nucleosides, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in 11 human leukocyte DNA samples were 16.2 +/- 5.2, 11.1 +/- 5.8, and 8.6 +/- 9.1 (mean +/- S.D.) in 10(8) normal nucleotides, respectively, starting from 30 mug of DNA or 1-1.5 mL of blood, and all the relative standard deviations were within 10%. An aliquot equivalent to 6 mug of DNA hydrolysate was used for analysis by this nanoLC-NSI/MS/MS. Thus, this highly sensitive and specific nanoLC-NSI/MS/MS method is suitable for accurate quantification of the three lipid peroxidation-derived etheno DNA adducts as noninvasive biomarkers in clinical studies for cancer risk assessment and for evaluation of preventive agents.

High Excretion of Etheno Adducts in Liver Fluke–Infected Patients: Protection by Praziquantel against DNA Damage

Chronic infection by Opisthorchis viverrini (OV) is a strong risk factor for developing cholangiocarcinoma (CCA). To clarify the involvement of oxidative stress and lipid peroxidation (LPO)-derived DNA damage, the excretion of LPO-derived etheno DNA adducts was measured in urine samples collected from healthy volunteers and OV-infected Thai subjects. 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) and 3,N(4)-etheno-2'-deoxycytidine (epsilondC) levels were quantified by immunoprecipitation/high-performance liquid chromatography/fluorescence detection and (32)P-postlabeling TLC. Excreted etheno adduct levels were related to indicators of inflammatory conditions [malondialdehyde (MDA) and nitrate/nitrite levels in urine and plasma alkaline phosphatase (ALP) activity]. Mean epsilondA and epsilondC levels were 3 to 4 times higher in urine of OV-infected patients; MDA, nitrate/nitrite, and ALP were also increased up to 2-fold. MDA and ALP were positively related to epsilondA excretion. Two months after a single dose of the antiparasitic drug Praziquantel, epsilondA and epsilondC concentrations in urine of OV-infected subjects were decreased; MDA, nitrate/nitrite, and ALP were concomitantly lowered. We conclude that chronic OV infection through oxidative/nitrative stress leads to increased urinary excretion of the etheno-bridged deoxyribonucleosides, reflecting high LPO-derived DNA damage in vivo. These promutagenic DNA etheno adducts in bile duct epithelial cells may increase the risk of OV-infected patients to later develop CCA. Urinary epsilondA and epsilondC levels should be explored (a) as noninvasive risk markers for developing opisthorchiasis-related CCA and (b) as promising biomarkers to assess the efficacy of preventive and therapeutic interventions.

Increased urinary 1, N6-ethenodeoxyadenosine and 3, N4-ethenodeoxycytidine excretion in thalassemia patients: Markers for lipid peroxidation-induced DNA damage

Thalassemic diseases including homozygous β-thalassemia and β-thalassemia/Hb E (β-Thal/Hb E) are prevalent in Southeast Asia. Iron overload is a common complication in β-thalassemia patients which induces intracellular oxidative stress and lipid peroxidation (LPO). LPO end products generate miscoding etheno adducts in DNA which after their repair are excreted in urine. We investigated whether urinary levels of 1, N 6 -ethenodeoxyadenosine (εdA) and 3, N 4 -ethenodeoxycytidine (εdC) can serve as putative cancer risk markers in β-Thal/Hb E patients. εdA and εdC levels were assayed in collected urine samples by immunoprecipitation-HPLC-fluorescence and 32P-postlabeling TLC, respectively. Mean εdA (fmol/µmol creatinine) levels in urine of β-Thal/Hb E patients ranged from 4.8 to 120.4 (33.8 ± 3.9; n = 37) and were 8.7 times higher compared to asymptomatic controls (1.4–13.8; 3.9 ± 0.8; n = 20). The respective εdC levels ranged from 0.15 to 32.5 (5.2 ± 1.3; n = 37) and were increased some 13 times over controls (0.04–1.2; 0.4 ± 0.7; n = 20). εdC levels were correlated positively with NTBI ( r = 0.517; P = 0.002), whereas εdA showed only a trend ( r = 0.257; P = 0.124). We conclude that the strongly increased urinary excretion of etheno adducts indicates elevated LPO-induced DNA damage in internal organs such as the liver. These highly promutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.

Risk factors and mechanisms of hepatocarcinogenesis with special emphasis on alcohol and oxidative stress

Hepatocellular cancer is the fifth most frequent cancer in men and the eighth in women worldwide. Established risk factors are chronic hepatitis B and C infection, chronic heavy alcohol consumption, obesity and type 2 diabetes, tobacco use, use of oral contraceptives, and aflatoxin-contaminated food. Almost 90% of all hepatocellular carcinomas develop in cirrhotic livers. In Western countries, attributable risks are highest for cirrhosis due to chronic alcohol abuse and viral hepatitis B and C infection. Among those with alcoholic cirrhosis, the annual incidence of hepatocellular cancer is 1-2%. An important mechanism implicated in alcohol-related hepatocarcinogenesis is oxidative stress from alcohol metabolism, inflammation, and increased iron storage. Ethanol-induced cytochrome P-450 2E1 produces various reactive oxygen species, leading to the formation of lipid peroxides such as 4-hydroxy-nonenal. Furthermore, alcohol impairs the antioxidant defense system, resulting in mitochondrial damage and apoptosis. Chronic alcohol exposure elicits hepatocyte hyperregeneration due to the activation of survival factors and interference with retinoid metabolism. Direct DNA damage results from acetaldehyde, which can bind to DNA, inhibit DNA repair systems, and lead to the formation of carcinogenic exocyclic DNA etheno adducts. Finally, chronic alcohol abuse interferes with methyl group transfer and may thereby alter gene expression.

Oxidative and alkylating damage in DNA.

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.

Novel 1,N(6)-etheno-2'-deoxyadenosine adducts from lipid peroxidation products.

trans,trans-2,4-Decadienal (DDE) is a widespread alpha, beta-unsaturated aldehyde found, for example, in food, water, and environmental pollutants. DDE is also endogenously generated as a breakdown product of lipid peroxidation in cell membranes. In the work presented here, the reaction of DDE with 2'-deoxyadenosine (dAdo) was investigated in an effort to assess its possible DNA damage potential. Besides 1,N(6)-etheno-2'-deoxyadenosine and two products, namely, 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1,2,3-octanetriol (adduct I) and 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1,2-heptanediol (adduct II), previously described by our group, two novel etheno adducts were identified. Thus, 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1-hexanol (adduct III) and 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-2,3-epoxy-1-octanol (adduct IV) were isolated by reverse-phase high-performance liquid chromatography and characterized on the basis of extensive spectroscopic measurements. The formation of the adducts is likely to involve initial DDE oxidation followed by generation of reactive intermediates such as diepoxides, epoxides, and/or hydroperoxides. The subsequent reaction of the latter oxidation products with dAdo will give rise to the four described adducts. We also demonstrated here that upon oxidation, DDE reacts with calf thymus DNA, producing the four dAdo adducts. Interestingly, two of them are the expected products arising from the reaction of dAdo with 4-hydroxy-trans-2-nonenal (HNE) and trans-2-octenal, two other important breakdown lipid peroxidation products. The reactivity of DDE with DNA is lower than that of the latter aldehydes. However, DDE produced a wider variety of adducts. The characterization of the different DNA-etheno adducts and the determination of the mechanism of formation are of great importance for a better understanding of the deleterious biological effects associated with this class of compounds.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch.

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (epsilonA) and 3,N4-ethenocytosine (epsilonC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises epsilonA. The present work is directed toward identifying and purifying the human glycosylase activity releasing epsilonC. HeLa cells were subjected to multiple steps of column chromatography, including two epsilonC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the epsilonC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having epsilonC-DNA glycosylase activity. In addition, oligonucleotides containing epsilonC.G or G/T(U), could compete for epsilonC protein binding, further indicating that the epsilonC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for epsilonC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.

DNA adducts of 2,3-epoxy-4-hydroxynonanal: detection of 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine and 1,N6-ethenoadenine by gas chromatography/negative ion chemical ionization/mass spectrometry.

2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxidation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyunsaturated fatty acids. EH is mutagenic and tumorigenic and capable of modifying DNA bases forming etheno adducts in vitro. Recent studies showed that etheno adducts are present in tissue DNA of humans and untreated rodents, suggesting a potential endogenous role of EH in their formation. A sensitive assay is needed so we can determine whether EH is involved in etheno adduct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative ion chemical ionization/mass spectrometry assay for the analysis of 1, N6-ethenoadenine (epsilonAde) and 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine (DHH-epsilonAde) in DNA; both are products from the reaction of adenine with EH. The assay entails the following sequence of steps: (1) addition of [15N5]epsilonAde and [15N5]DHH-epsilonAde to DNA as internal standards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid phase extraction (SPE), (4) derivatization by pentafluorobenzylation (PFB), (5) separation of PFB-epsilonAde and PFB-DHH-epsilonAde on a Si SPE column, (6) acetonide (ACT) formation of PFB-DHH-epsilonAde, and (7) GC/MS analysis with selective ion monitoring (SIM). The limit of detection by on-column injection for PFB-epsilonAde monitoring of the (M - PFB)- ion at m/z 158 was 30 amol and for ACT-PFB-DHH-epsilonAde monitoring of the (M - PFB)- ion at m/z 328 was 0.4 fmol; the detection limits for the entire assay were 6.3 fmol for epsilonAde and 36 fmol for DHH-epsilonAde. In calf thymus DNA modified with EH at 37 degreesC for 50 h, both epsilonAde and DHH-epsilonAde were detected at high levels by this method, 4.5 +/- 0.7 and 90.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also verified by HPLC fluorescence analysis, indicating that EH extensively reacts with adenine in DNA, forming etheno adducts. The high sensitivity of the assay suggests that it may be used in the analysis of ethenoadenine adducts in vivo.

Formation of DNA etheno adducts in rodents and humans and their role in carcinogenesis.

Ethenobases are exocyclic adducts formed with DNA by some environmental carcinogens such as vinyl chloride or urethane. In the last few years, they have received a renewed interest due to the development of sensitive techniques of analysis that made it possible to measure their formation in vivo. This minireview summarizes the information gained recently from the work of several laboratories, including ours. Increased levels of DNA etheno adducts have been measured in target tissues from rodents exposed to vinyl chloride or urethane. Hepatic tumours caused by exposure to vinyl chloride in humans and in rats and lung tumours induced by urethane in mice exhibit base pair substitution mutations in the ras and p53 genes which seem to be exposure-specific and consistent with the promutagenic properties of ethenobases. Background levels of etheno adducts have been detected in DNA from non-exposed humans or animals, pointing to an alternative, endogenous pathway of formation. This background may be affected by dietary factors. It could arise from the reaction of trans-4-hydroxy-2-nonenal (or its epoxide 2,3-epoxy-4-hydroxynonanal), a lipid peroxidation product, with nucleic acid bases. Elevated levels of etheno adducts are found in hepatic DNA from humans and rodents with genetic predisposition to oxidative stress and lipid peroxidation in the liver, and with an associated increased risk of liver cancer. These data suggest that DNA ethenobases could serve as new biomarkers of oxidative stress/lipid peroxidation.

Etheno-DNA base adducts as tools in human cancer aetiology and chemoprevention.

Etheno adducts in DNA are formed from the carcinogens vinyl chloride and urethane, and also from products of lipid peroxidation (LPO), such as trans-4-hydroxy-2-nonenal. Using an ultrasensitive detection method, the formation of etheno-DNA adducts in the liver was demonstrated in LEC rats (a strain with hereditary abnormal copper metabolism) that develop hepatitis and hepatocellular carcinoma. Wilson's disease and primary haemochromatosis are human genetic disorders that cause copper or iron accumulation resulting in a high risk for primary liver cancers. Levels of etheno adducts were also significantly elevated in the liver of these patients. In a group of male and female volunteers kept on a controlled diet, the effect of dietary fatty acid composition on the endogenous formation of lipid peroxidation-derived DNA adducts was determined in DNA from white blood cells. Dietary omega-6-polyunsaturated fatty acids greatly increased LPO-derived etheno-DNA adducts in vivo, in females. Thus, exocyclic DNA adducts are promising biomarkers for elucidating the effect of dietary fat intake, oxidative stress and protective dietary antioxidants on endogenous DNA damage and thus may provide a possible mechanistic link with elevated risk for diet-related cancers.

DNA damage, and repair in mutagenesis and carcinogenesis: implications of structure-activity relationships for cross-species extrapolation

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD 50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD 50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters ( s-values, 0 6/ N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD 50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the M exr−/M exr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD 50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, k c, reaction at a critical nucleophilic strength, n c. For alkylating agents k c can be expressed as the second-order rate constant for hydrolysis, k H 2O , and the substrate constant s: k H 2O TD 50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, a c, for 2 years exposure of the rodents. The TD 50 in mmol/kg × day) could then be written: T−fsD 50 ∗= a c×λ k H 2O ×10 nc s . This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most S N2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be improved by considering the distribution of dose in the body. In the present analysis, direct-acting alkylators were studied. It is evident that dosimetry and reaction-kinetic characterization of genotoxic metabolites would render it possible to predict the potency of precarcinogens as well.