The mechanism by which environmentally prevalent N-nitrosodiethanolamine (NDELA) and related 2-hydroxyethyl- or other beta-oxidized nitrosamines initiate the carcinogenic process has remained obscure. (32)P-Postlabeling assays for the pH sensitive glyoxal-deoxyguanosine (gdG) and the O(6)-2-hydroxyethyldeoxyguanosine (OHEdG) DNA adducts have been developed as probes in this mechanistic investigation and used in both in vitro and in vivo experiments. The ready cleavage of the glyoxal fragment from gdG at pH 7 and greater has required methods of optimization in order to achieve a detection limit of 0.05 micromol/mol of DNA. Nuclease P1 treatment enhances the detection of gdG adducts but does not increase the detection limit for OHEdG. For OHEdG, best results were achieved using fraction collection from HPLC (0.3 micromol/mol of DNA). Using radiochemical methods, both adducts could be detected either by HPLC or 2D TLC. NDELA, N-nitrosomorpholine (NMOR), N-nitrosomethyethanolamine (NMELA), and N-nitrosoethylethanolamine (NEELA) all produce both gdG and OHEdG adducts in rat liver DNA in vivo and are called bident carcinogens because fragments from both chains of the nitrosamine are incorporated into DNA. N-Nitroso-2-hydroxymorpholine (NHMOR), a metabolite of NDELA and NMOR, generates gdG in DNA in vitro and in vivo. gdG DNA adducts were found in the range 1.1-6.5 micromol/mol of DNA. OHEdG DNA adducts were produced from equimolar amounts of nitrosamines in rat liver in vivo over the range 4-25 micromol/mol of DNA and in the order NMELA > NEELA > NDELA > NMOR. Deuterated isotopomers of NDELA showed a marked isotope effect on DNA OHEdG adduct formation. alpha-Deuteration markedly decreased OHEdG adduct formation while beta-deuteration had the opposite effect. These data support the hypothesis that NDELA and related nitrosamines are activated by both enzyme mediated alpha-hydroxylation and beta-oxidation. The formation of OHEdG adducts from NDELA requires alpha-hydroxylation of the 2-hydroxyethyl chain, and formation of gdG necessitates a beta-oxidation as well. The bident nature of these carcinogens may explain why they are relatively potent carcinogens despite the fact that major proportions of doses are excreted unchanged.

In a previous study we demonstrated the ability of dopamine (DA) to stimulate phospholipid methylation (PLM) via a novel mechanism involving the D4 dopamine receptor (D4R) in which single-carbon folates appeared to be the primary source of methyl groups. To further understand the relationship between D4R-mediated PLM and folate metabolism, we examined the effect of several folate pathway interventions on the level of basal and DA-stimulated incorporation of (iv) Growth in nucleoside-free media. 31 P-NMR was also used to follow DA-induced changes in cell phospholipid composition. MTHFS overexpression and 5-formylTHHF treatment, both of which lower 5-methylTHF levels, each reduced basal PLM and its stimulation by DA. In contrast, 5-formylTHF, which increases 5-methylTHF, caused a dosedependent increase in both basal and DA-stimulated PLM. Growth in nucleoside-free media caused time-dependent changes in PLM, which were due to the absence of purine nucleosides. While basal PLM was maintained at a reduced level, DA-stimulated PLM was initially increased followed by a later decrease. Together, these ®ndings indicate a close functional relationship between single-carbon folate metabolism and DA-stimulated PLM, consistent with a role for 5-methylTHF as the methyl donor for the D4R-mediated process. Keywords: D4 dopamine receptors, folic acid metabolism, 5-methyltetrahydrofolate, phospholipid methylation, purine metabolism, schizophrenia.

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Objectives The impact of the COVID-19 pandemic on breastfeeding practices in low and middle-income countries is not well understood. Modifications in breastfeeding guidelines and delivery platforms for breastfeeding education are hypothesized to have affected breastfeeding practices during the COVID-19 pandemic. We aimed to understand the experiences with perinatal care, breastfeeding education and practice among mothers who delivered infants during the COVID-19 pandemic. Methods We conducted key informant interviews among 35 mothers with deliveries since March 2020 and 10 healthcare workers (HCW) from two public health facilities in Naivasha, Kenya. Results Mothers described COVID-related income loss and lack of support from family and friends as a worse challenge to practicing EBF as they wished or planned. While mothers noted that HCWs provided quality care and breastfeeding messaging, one-on-one perinatal breastfeeding education was cited to be less frequent than before the pandemic due to altered conditions in health facilities and COVID safety protocols. Knowledge among mothers about the safety of breastfeeding in the context of COVID was limited, with few key informants reporting of specific receipt of information such as COVID transmission through human milk and the safety of nursing during a COVID infection. Mothers stated that some HCW messages emphasized the immunologic importance of BF. COVID restrictions limited or prevented familial support at facilities and home, causing stress and fatigue for mothers. In some cases, mothers noted income loss due to furloughs and layoffs, time spent seeking new means of employment, and food insecurity as causes for perceived milk insufficiency, which was, in turn, connected to introducing weaning foods and liquids before six months. Conclusions The COVID-19 pandemic created changes to the perinatal experience for mothers. While messages the importance of practicing exclusive breastfeeding were provided, altered HCW education delivery methods, social support and food insecurity limit EBF practices in for mothers in this context. Mothers lacked consistent knowledge about the safety of breastfeeding in the context of COVID-19. Funding Sources National Institutes of Health Fogarty International Center.

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