**Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats**

**Leticia Díez-Quijada 1, Concepción Medrano-Padial 1, María Llana-Ruiz-Cabello 1, Giorgiana M. Cătunescu 2, Rosario Moyano 3, Maria A. Risalde 4,5, Ana M. Cameán 1,\* and Ángeles Jos <sup>1</sup>**


Received: 8 April 2020; Accepted: 23 May 2020; Published: 26 May 2020

**Abstract:** Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

**Keywords:** in vivo; genotoxicity; cylindrospermopsin; microcystin-LR; micronucleus; comet assay; enzyme-modified comet assay; rats

**Key Contribution:** CYN/MC-LR combinations induced in rats increases MN formation in bone marrow. No DNA strand breaks were induced and the DNA was not oxidatively damaged as detected by the comet assays.
