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. 2022 Jul;31(5):860-872.
doi: 10.1007/s10646-022-02551-5. Epub 2022 May 17.

The reproductive effects of the cancer chemotherapy agent, Carmofur, on Daphnia magna are mediated by its metabolite, 5-Fluorouracil

Emily E Gessner #  1 , Manav H Shah #  1 , Bricen N Ghent  1 , Nathaniel E Westbrook  1 , Peter van den Hurk  1 , William S Baldwin  2
Affiliations

The reproductive effects of the cancer chemotherapy agent, Carmofur, on Daphnia magna are mediated by its metabolite, 5-Fluorouracil

Emily E Gessner et al. Ecotoxicology. 2022 Jul.

Abstract

Carmofur is an antineoplastic agent that inhibits ceramidase, a key enzyme in the sphingolipid pathway. Previous research suggests carmofur represses reproductive maturity in Daphnia magna. The purpose of this experiment was to confirm carmofur's effects on fecundity and reproductive maturity over two generations. A chronic toxicity test found reproductive maturity was delayed from 9 to 19 days by 0.80 μM carmofur with a 99.7% drop in reproduction, probably caused by delayed ovarian development. Second generation effects were even greater with 0% reproductive success at 0.40 μM. To our surprise, carmofur was not measured in the media by HPLC 24 h after exposure. Previous research indicated that carmofur is unstable in water and hydrolyzed into 5-fluorouracil (5-FU). Therefore, the chronic toxicity study was repeated with 5-FU and similar effects on reproductive maturity were observed at similar concentrations despite very different acute toxicities (48 h carmofur LC50 = 1.93 μM; 5-FU LC50 = 207 μM). 5-FU delayed reproductive maturity from 9 to 21 days with a 71.12% drop in reproduction at 0.80 μM and greater effects in the 2nd generation similar to carmofur. 5-FU was found stable in aquatic media and HPLC confirmed 5-FU was hydrolyzed from carmofur within 24 h. In conclusion, carmofur and 5-FU reduce fecundity because they delay reproductive maturity and ovarian development in Daphnia magna. We conclude that the reproductive effects observed after carmofur treatment are primarily mediated by its breakdown product, 5-FU. This further underscores the importance of measuring chemical concentrations and evaluating chemical metabolism and decomposition when determining toxicity, especially of chemotherapeutic agents.Clinical trials registration Not applicable.

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Conflict of interest statement

Conflicts of Interest

The authors have no conflicts of interest to declare

Figures

Figure 1.
Figure 1.. Carmofur decreases fecundity in a concentration-dependent manner.
A chronic toxicity test was performed and neonates per daphnid were determined for 21-days. Statistical analysis was performed by one-way ANOVA with * p < 0.01; ** p < 0.001 (n = 13). (a) indicates all exposure groups are significantly different from the control (UT), (b) indicates all exposure groups 0.10 μM and greater are different from the control and (c) indicates exposure groups 0.20 μM and greater are significantly different from the control.
Figure 2.
Figure 2.. Carmofur reduced reproduction in a concentration-dependent manner in the first generation.
A chronic toxicity test was performed and (A) the total number of reproductive daphnids, (B) mean age of first showing ovaries, (C) mean number of broods per daphnid, (D) mean age at first brood, and (E) the mean number of offspring per brood was determined. Statistical analysis was performed by one-way ANOVA with * p < 0.05 , ** p < 0.0005; *** p < 0.0001 compared to the control (n = 13). Data are shown as mean ± SEM (n= 13).
Figure 3.
Figure 3.. Total number of neonates produced decreases as carmofur concentration (μM) increases in the second generation.
A second generation chronic toxicity test was performed and the number of neonates produced per daphnid was recorded for 21-days. Statistical analysis was performed via one-way ANOVA. *p < 0.01 and **p < 0.001. (a) indicates all exposure groups are significantly different from the control and (b) indicates all exposure groups 0.10 μM and greater are different from the control.
Figure 4.
Figure 4.. There was an inverse relation between reproductive success and carmofur concentration in second generation chronic toxicity tests.
(A) The total number of reproductive daphnids, (B) mean age of first showing ovaries, (C) mean number of broods released per daphnid, (D) mean age at first reproduction, and (E) mean number of offspring per brood were determined. Statistical analysis was performed by one-way ANOVA with * p < 0.05 , ** p < 0.0005; *** p < 0.0001 compared to the control. * indicates p <0.05; ** indicates p < 0.0001 (n = 10). Data are shown as mean ± SEM (n = 10).
Figure 5:
Figure 5:. Acute Toxicity of Carmofur and 5-FU.
An acute toxicity test was conducted on Daphnia magna comparing carmofur and 5-FU over 24 and 48 hours of exposure. LC50’s were determined using GraphPad Prism 7.0 as described in the Materials and Methods.
Figure 6.
Figure 6.. 5-Fluorouracil reduces the fecundity of Daphnia magna in a concentration-dependent manner.
A chronic toxicity test was performed and neonates per daphnid were determined for 21-days. Statistical analysis was performed by one-way ANOVA with * p < 0.001; ** p < 0.0001 (n = 12). (a) indicates all exposure groups are significantly different from the control, (b) indicates all exposure groups different from the control with a p < .0001 significance, except for 0.2 μM which is different from the control with a p < 0.001 significance.
Figure 7.
Figure 7.. 5-Fluorouracil reduced reproduction in Daphnia magna in a concentration-dependent manner in the first generation.
A chronic toxicity test was performed and (A) the total number of reproductive daphnids, (B) the mean age of Daphnia magna first showing eggs, (C) the mean number of broods released per Daphnia (D) the mean age at first brood, and (E) the mean number of offspring per brood were determined. Statistical Analysis was performed by one-way ANOVA and ** indicates a p-value < 0.0001 (n = 12).
Figure 8:
Figure 8:. 5-Fluorouracil reduced fecundity of Daphnia magna in a concentration-dependent manner in the second generation.
A second generation chronic toxicity test was performed and neonates per daphnid were detennined for 21-days. Statistical Analysis was performed by one-way ANOVA with * indicating p < .05, ** indicating p < .01, and *** indicating p < 0.0001 between the control and the treated groups.
Figure 9.
Figure 9.. 5-Fluorouracil reduced reproduction of Daphnia magna in the second generation
A second generation chronic toxicity test was performed and (A) the total number of reproductive daphnids, (B) the mean age of Daphnia magna first showing eggs, (C) the mean number of broods released per Daphnia, (D) the mean age at first brood release, and (E) the mean number of offspring per brood were determined. Statistical Analysis was performed by one-way ANOVA; * p < .001; ** p < 0.0001 (n = 10).
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