Transgenerational Reproductive and Developmental Toxicity Induced by N-Nitrosodimethylamine and Its Metabolite Formaldehyde in Drosophila melanogaster

Abstract

N-Nitrosodimethylamine (NDMA) is a known water disinfection byproduct (DBP) characterized as a potent hepatotoxin, promutagen, and probable human carcinogen; this is because of the metabolites associated with its biotransformation. The metabolism of NDMA produces formaldehyde, another alkylating agent and DBP. Both compounds are generated from natural and anthropogenic sources, but the safety restrictions applied to NDMA do not extend to the uses of formaldehyde. Hence, potential health and ecological risks are of concern. Due to limited information on the long-term effects of exposure to these compounds at environmentally relevant concentrations, this work aimed to compare the transgenerational reproductive and developmental toxicity of separate exposures to NDMA or its metabolite formaldehyde in Drosophila melanogaster over four generations. The parental flies were fed NDMA or formaldehyde (1.19E-06 to 5 mM) for 48 h during the third larval instar. Subsequent offspring (F1-F3) were grown under compound-free conditions. In the parental generation, both exposures modified the time to emergence and reduced the number of progenies. NDMA, but not formaldehyde, was lethal, affected fertility, and weakly induced malformations. In the next generations, both exposures induced malformed flies and modified the number of offspring. Reproductive toxicity and malformations were maintained for at least three generations, suggesting that detrimental effects could extend to unexposed offspring. This is the first study reporting the associated individual transgenerational effects on reproduction and development between NDMA and its metabolite formaldehyde in D. melanogaster, highlighting the relevance of evaluating multiple generations to accurately determine the health and environmental risks of pollutants.

Keywords: DBPs; Drosophila melanogaster; development; dimethylnitrosamine (DMN); fecundity; fertility; formalin; morphological alterations; parental exposure; reproduction.

FIGURE 1

Toxicity of NDMA in D. melanogaster . Third‐instar larvae were exposed during 48 h to different concentrations (0.313, 0.625, 1.04, 1.25, 1.5, and 5 mM) of NDMA. The LC50 was estimated at 1.18 mM via the AAT Bioquest Calculator considering the loss of survival (mean ± SEM, n = 3, two vials per concentration, 100 larvae per vial). A negative correlation was obtained between survival and NDMA concentration ( $\widehat{r}$ _Pearson = −0.89, p < 7.08E−03).

FIGURE 2

Time to emergence of D. melanogaster exposed to NDMA or formaldehyde. (A)–(C) refer to NDMA, and (D)–(F) allude to formaldehyde exposures. (A–D), (B–E), and (C–F) show the proportion of flies that emerged per day (13, 16, and 19) at low, medium, and high concentrations, respectively (mean ± SEM, n = 3, two vials per concentration, 100 larvae per vial); *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the control (Mann–Whitney U test).

FIGURE 3

Modifications in the reproductive capacity of D. melanogaster following exposure to NDMA or formaldehyde in the parental generation (P) and subsequent unexposed offspring (F1–F3). (A) and (C) refer to NDMA, (B) and (D) allude to formaldehyde exposures. (A) and (B) show effects on fertility and (C) and (D) on fecundity (number of offspring) across generations. All values are presented as a corrected index, where the data above the control are positive and below negative (n = 3, five vials per concentration, one female and one male per vial). Significant differences from the control are designated as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (proportion Z‐test for fertility; one‐way ANOVA or the Kruskal–Wallis test for fecundity).

FIGURE 4

Morphological alterations observed in D. melanogaster flies (P generation) exposed to NDMA or its metabolite formaldehyde and their untreated offspring (F1–F3). The black arrows indicate abnormalities in the corporal structures of Drosophila adults. * designates a fly obtained specifically from the F3 generation after parental exposure to formaldehyde. ** shows malformed phenotypes derived from NDMA‐exposed parental flies also found in the F3 generation of formaldehyde‐treated parental flies.