Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2'-Deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2'-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2'-deoxyinosine 9 was prepared from 2'-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-15N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-ß-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

The enemy at the gates? DNA adducts as biomarkers of exposure to exogenous and endogenous genotoxic agents

Genomic DNA is under continuous assault by various chemical species produced by normal cellular metabolism. In addition, exposure to exogenous agents adds further insult. Modification of DNA by chemical carcinogens has long been recognized as an early event in carcinogenesis and many DNA adducts have been characterized. There appears to be great value in using DNA adducts as markers of exposure to genotoxic (i.e. DNA-damaging) agents and some may be even more useful as indicators of risk of disease. Studies of the relationship between aflatoxin exposure and liver cancer have illustrated particularly well the advantages of using specific DNA adducts and other biomarkers, not only to better characteristic the risk factors, but also as endpoints in intervention studies. DNA adducts of endogenous genotoxins such as malondialdehyde and nitrosated glycine are particularly informative in studies of the effects of diet on cancer risk. DNA adducts may also be useful in identifying no-exposure levels in risk assessment of low-level environmental exposures such as as 1,3-butadiene (BD)

Potassium diazoacetate-induced p53 mutations in vitro in relation to formation of O6-carboxymethyl- and O6-methyl-20-deoxyguanosine DNA adducts: relevance for gastrointestinal cancer

Nitrosated glycine derivatives react with DNA to form O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG) and O6-methyl-2'-deoxyguanosine (O6-MedG) adducts concurrently. O6-CMdG is not repaired by O6-alkylguanine alkyltransferases and might be expected to lead to mutations via a similar mechanism to O6-MedG. Potassium diazoacetate (KDA) is a stable form of nitrosated glycine and its ability to induce mutations in the p53 gene in a functional yeast assay was studied. Treatment of a plasmid containing the human p53 cDNA sequence with KDA afforded readily detectable levels of O6-CMdG and O6-MedG. The treated plasmid was used to transform yeast cells and coloured colonies harbouring a p53 sequence with functional mutations were detected. Recovery of the mutated plasmids followed by DNA sequencing enabled the mutation spectrum of KDA to be characterised. The most common mutations induced by KDA were substitutions with >50% occurring at GC base pairs. In contrast to the methylating agent methylnitrosourea which gives predominantly (>80%) GCAT transitions, KDA produced almost equal amounts of transitions (GCAT) and transversions (GCTA and ATTA). This difference is probably due to a different mode of base mispairing for O6-CMdG compared with O6-MedG. The pattern of mutations induced by KDA was very similar to the patterns observed in mutated p53 in human gastrointestinal tract tumours. These results are consistent with the hypothesis that nitrosation of glycine (or glycine derivatives) may contribute to characteristic human p53 mutation profiles. This conclusion is borne out by recent observations that O6-CMdG is present in human DNA both from blood and exfoliated colorectal cells and is consistent with recent epidemiological studies that have concluded that endogenous nitrosation arising from red meat consumption is related to an increased risk of colorectal cancer

Red Meat Enhances the Colonic Formation of the DNA Adduct O6-Carboxymethyl Guanine: Implications for Colorectal Cancer Risk

Red meat is associated with increased risk of colorectal cancer and increases the endogenous formation of N-nitrosocompounds (NOC). To investigate the genotoxic effects of NOC arising from red meat consumption, human volunteers were fed high (420 g) red meat, vegetarian, and high red meat, highfiber diets for 15 days in a randomized crossover design while living in a volunteer suite, where food was carefully controlled and all specimens were collected. In 21 volunteers, there was a consistent and significant (P < 0.0001) increase in endogenous formation of NOC with the red meat diet compared with the vegetarian diet as measured by apparent total NOC (ATNC) in feces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O6-carboxymethyl guanine (O6CMG) was significantly (P < 0.001) higher on the high red meat diet. In 13 volunteers, levels were intermediate on the high-fiber, high red meat diet. Fecal ATNC were positively correlated with the percentage of cells staining positive for O6CMG (r2 = 0.56, P = 0.011). The presence of O6CMG was also shown in intact small intestine from rats treated with the N-nitrosopeptide N-acetyl-NV-prolyl-NV-nitrosoglycine and in HT-29 cells treated with diazoacetate. This study has shown that fecal NOC arising from red meat include direct acting diazopeptides or N-nitrosopeptides able to form alkylating DNA adducts in the colon. As these O6CMG adducts are not repaired, and if other related adducts are formed and not repaired, this may explain the association of red meat with colorectal cancer. (Cancer Res 2006; 66(3): 1859-65)

Deleterious effects of a cafeteria diet on the livers of non-obese rats

Rats were fed from weaning on three diets. Those fed a cafeteria diet had livers that were enlarged and abnormal by visual inspection. The rats themselves appeared healthy, had a normal growth rate and were not significantly different in weight from control animals. Histological examination revealed the livers of these rats to be rich in lipids and glycogen. Liver function tests showed a depressed level of alanine transaminase and an abnormal HDL: LDL. Dietary lipids generate free radicals which can interact with, and damage, DNA. However, when DNA was extracted from the livers and examined for the presence of the adduct M1-dG, there were no significant differences in adduct levels in livers from animals fed any of the diets. We conclude that the cafeteria diet can have long term adverse effects on liver function even though overt measures of health may be unimpaired, body mass maintained within normal limits, and liver DNA not adversely affected

Lobe-specific increases in malondialdehyde DNA adduct formation in the livers of mice following infection with Helicobacter hepaticus

Helicobacter hepaticus infection is associated with chronic hepatitis and the development of liver tumours in mice. The underlying mechanism of this liver carcinogenesis is not clear but the oxidative stress associated with H. hepaticus infection may result in induction of lipid peroxidation and the generation of malondialdehyde. Malondialdehyde can react with deoxyguanosine in DNA resulting in the formation of the cyclic pyrimidopurinone N-1,N-2 malondialdehyde-deoxyguanosine (M(1)dG) adduct. This adduct has the potential to cause mutations that may ultimately lead to liver carcinogenesis. The objective of this study was to determine the control and infection-related levels of M(1)dG in the liver DNA of mice over time, using an immunoslot-blot procedure. The level of M(1)dG in control A/J mouse livers at 3, 6, 9 and 12 months averaged 37.5, 36.6, 24.8 and 30.1 adducts per 10(8) nucleotides, respectively. Higher levels of M(1)dG were detected in the liver DNA of H. hepaticus infected A/JCr mice, with levels averaging 40.7, 47.0, 42.5 and 52.5 adducts per 10(8) nucleotides at 3, 6, 9 and 12 months, respectively. There was a significant age dependent increase in the level of M(1)dG in the caudate and median lobes of the A/JCr mice relative to control mice. A lobe specific distribution of the M(1)dG adduct in both infected and control mice was noted, with the left lobe showing the lowest level of the adduct compared with the right and median lobes at all time points. In a separate series of mice experimentally infected with H. hepaticus, levels of 8-hydroxy-deoxyguanosine were significantly greater in the median compared with the left lobe at 12 weeks after treatment. In conclusion, these results suggest that M(1)dG occurs as a result of oxidative stress associated with H. hepaticus infection of mice, and may contribute to liver carcinogenesis in this model

Chemical genetics

This review covers some of the highlights in the field of chemical genetics published during 2004. A key enabling methodology in the field is the availability of diverse libraries of small molecules provided by combinatorial chemistry, and notable advances in this area are included. As the chemical genetic approach becomes more widely established the number of new biological pathways targeted by novel small molecules increases, and significant discoveries made during the year are summarised. Of particular note during 2004 was the publication of a series of review and commentary articles on the theme of ‘‘chemical space’’—a concept central to chemical genetics