Cylindrospermopsin-microcystin-LR combinations may induce genotoxic and histopathological damage in rats

toxins Article 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. Cameán1,* and Ángeles Jos 1 1Area of Toxicology, Faculty of Pharmacy, University of Sevilla, Profesor García González n2, 41012 Sevilla, Spain; [email protected] (L.D.-Q.); [email protected] (C.M.-P.); [email protected] (M.L.-R.-C.); [email protected] (Á.J.) 2University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăs ,tur 3-5, 400372 Cluj-Napoca, Romania; [email protected]o 3 Department of Pharmacology, Toxicology and Legal and Forensic Medicine, Faculty of Veterinary Medicine, University of Córdoba, Campus de Rabanales, 14014 Córdoba, Spain; [email protected] 4Animal Pathology Department. Faculty of Veterinary Medicine, University of Córdoba, Campus Universitario de Rabanales s/n, 14014 Cordoba, Spain; [email protected] 5 Instituto Maimonides de Investigaci ó n Biom é dica de C ó rdoba (IMIBIC)-Hospital Universitario Reina Sof í a de Córdoba-Universidad de Córdoba, Avenida Menendez Pidal s/n, 14006 Cordoba, Spain *Correspondence: [email protected]; Tel.: +34-954-556762 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 µ g 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. 1. Introduction Climate and nutrient changes are contributing to global eutrophication and global expansion of harmful algal blooms [ 1 ], including the proliferation of cyanobacterial blooms [ 2 ]. Cyanobacteria Toxins 2020,12, 348; doi:10.3390/toxins12060348 www.mdpi.com/journal/toxins Toxins 2020,12, 348 2 of 20 are producers of a broad group of secondary metabolites called cyanotoxins [ 3 ]. The main human exposure route to cyanotoxins is oral intake, primarily from drinking water. However, the consumption of contaminated food and dietary supplements with cyanotoxins cannot be disregarded, although some of them (such as cylindrospermopsin) have not been found in commercially-available blue-green algal supplements until recently [ 2 , 4 , 5 ]. The most studied cyanotoxins are microcystins (MCs) and cylindrospermopsin (CYN) as a consequence of their toxicity and wide distribution. The severity of these intoxications depends on several factors, such as their chemical structure, mechanisms of action, and concentrations of cyanotoxins involved [2]. MCs are cyclic heptapeptides that are mainly hepatotoxic [ 6 ], including as primary target organs the liver and kidney, as well as several secondary targets such as testes, ovaries, heart, lung, and the CNS; to date 246 MC congeners have been identified [ 7 ]. Microcystin-LR (MC-LR) is the most studied MC variant, because of its higher toxicity and wider distribution than other MC congeners [ 8 , 9 ]. Hepatotoxicity of MC-LR is mediated by the presence of organic anion transport polypeptides (OATPs) which are expressed mostly in the liver and are responsible for its uptake into the hepatocytes [ 10 ]. MC-LR is a specific inhibitor of protein serine/threonine phosphatases 1 and 2A (PP1, PP2A) [ 11 ]. It induces a hyperphosphorylation of cytoskeletal proteins affecting cell morphology and cellular adhesion, leading to necrosis [ 12 , 13 ]. Oxidative stress is another mechanism involved in MC toxicity [ 14 – 17 ]. Therefore, MC-LR could have carcinogenic or genotoxic properties because of its inhibitory action on PP1 and PP2A, acting as a liver tumor-promoter [ 18 – 20 ]. In fact, MC-LR was classified only as a possible human carcinogen (Group 2B) by the International Agency of Research on Cancer (IARC) [ 21 ], mainly because of the lack of sufficient evidence for its direct carcinogenicity in both humans and experimental animals [2]. The genotoxicity of pure MC-LR cyanotoxin has been widely studied and reviewed in the scientific literature [ 20 , 22 ]. Globally, contradictory results were reported following in vitro and in vivo experimental systems, and the mechanisms involved are not yet fully understood. Pioneering studies indicated that MC-LR damaged the DNA in the liver of male and female Swiss albino mice dependent upon the assayed dose and on the duration of exposure after intraperitoneal injection (i.p.) [ 23 ]. This effect was observed after just a single administration of a LD 50 dose of MC-LR [ 6 , 24 ]. Likewise, DNA damage was observed in the blood cells of Swiss albino mice after oral exposure to a single dose of MC-LR. Higher damage was found in different organs after i.p. injection [ 25 ]. Moreover, MC-LR induced a rapid increase of the amount of DNA in the tail of the comets and increased micronucleus (MN) frequencies in male mice injected i.p. [ 26 ] and in Balb/c mice exposed repeatedly to MC-LR for 30 d [ 27 ]. By contrast, other studies reported that MC-LR did not induce DNA damage in vivo . Thus, no DNA damage was observed in rat liver after a single intravenous administration (i.v.) of MC-LR [ 28 ], and negative results were observed in the MN assay in male transgenic mice after intragastric administration [29], and in male CBA mice after i.p. injection of MC-LR [19]. CYN is a stable tricyclic alkaloid consisting of a guanidine moiety combined with a hydroxymethyluracil [ 30 ], whose occurrence in aquatic systems is increasing. This cyanotoxin can be produced by different cyanobacterial genera such as Aphanizomenon, Cylindrospermopsis, Lyngbya, Oscilliatoria, Raphidiopsis and Umezaki, but Cylindrospermopsis raciborskii is its main producer [ 31 , 32 ]. CYN has cytotoxic activity and its effects on the liver and other organs have been proven [ 2 , 33 ]. The main mechanism of CYN toxicity is the inhibition of both protein and glutathione synthesis [ 34 – 36 ], although oxidative stress is also involved [ 37 ]. Several studies have shown that cytochrome P450 enzymes are necessary in the metabolic activation of CYN, and consequently in its toxicity [38,39]. At present, the mechanisms of pro-genotoxicity and potential carcinogenic activity of CYN (not yet classified by the IARC) are still not completely described and, thus, further investigations are needed. To this end, several in vivo studies in rodents were performed to clarify the genotoxicity of CYN. Thus, CYN induced DNA strand breaks (sb) in the liver of mice after i.p. administration [ 40 ]. Similarly, DNA damage was observed in the colon of mice after i.p. injection of the toxin, and both in the colon and bone marrow after oral (gavage) administration [ 41 ]. Dordevic et al. [ 42 ] demonstrated DNA damage Toxins 2020,12, 348 3 of 20 (comet assay) in the liver of rats exposed i.p. to CYN and to an extract of Cylindrospermopsis raciborskii. Recently, Diez-Quijada et al. [ 43 ] exposed rats to pure CYN (7.5–75.0 µ g/kg b.w) and performed a battery of assays consisting of MN in bone marrow, as well as the standard and modified comet assays in stomach, liver and blood. The DNA seemed to be damaged only in the bone marrow of rats regardless of concentration. By contrast, neither DNA strand breaks nor oxidative DNA damage was observed in the comet assays in any of the investigated tissues. Both MC-LR and CYN can be found at the same time in the environment and their simultaneous presence was previously described [ 44 – 46 ]. Thus, the European Food Safety Authority (EFSA) documented and stated the importance of studying the effects of their combined exposure [ 47 ]. A few studies dealt with the toxicological profile of CYN and MC-LR combinations. Thus, the in vitro assessments of the potential interactions of CYN and MC-LR are very scarce [ 48 – 51 ], and only two focus on their genotoxicity. Hercog et al. [ 48 ] described an induction of DNA sb by MN and comet assays at 24 h after the treatment of HepG2 cells with CYN/MC-LR combinations, but to a lesser extent for only CYN. The genomic instability detected by the cytokinesis block micronucleus assay was, however, comparable to the individual CYN. Recently, Diez-Quijada et al. [ 49 ] applied a battery of in vitro tests in several cell lines, including bacterial systems, and they described genotoxic effects only in the MN test, when the metabolic fraction S9 was used. To the best of our knowledge, no in vivo studies have been yet performed to assess the genotoxicity of CYN/MC-LR mixtures and they are necessary to elucidate the contradictory results obtained in vitro. Thus, according to the recommendations of the EFSA [ 52 ], the purpose of this research was to investigate, for the first time, the potential in vivo genotoxicity of CYN/MC-LR combinations in rats, as an experimental model, after oral administration (gavage) of relevant environmental concentrations. A combined MN—standard and modified comet assay was applied. The enzyme-modified comet assay was performed with Endonuclease-III (Endo-III) and Formamidopyrimidine glycosilase (Fpg) enzymes. Bone marrow was the selected tissue for the MN test, Organisation for Economic Co-operation and Development (OECD )474 [ 53 ], and stomach, liver OECD 489 [ 54 ] and blood cells for the standard and enzyme-modified comet assays. Additionally, the potential histopathological alterations were assessed in stomach and liver. 2. Results 2.1. Micronucleus Assay This assay was conducted following the recommendations of OECD guideline 474 [ 53 ], and it is especially relevant for assessing genotoxicity because, although they may vary among species, factors of in vivo metabolism, pharmacokinetics, and DNA repair processes are active and contribute to the responses. Its purpose is to identify cytogenetic damage which results in the formation of micronuclei (MN) containing lagging chromosome fragments or whole chromosomes. As positive control, ethylmethanesulfonate (EMS) was chosen according to this guideline. Results are measured as the polychromatic erythrocytes (PCE) out of total erythrocytes (normochromatic erythrocytes (NCE) +(PCE)), and the PCE/NCE ratios, which were calculated by counting 500 erythrocytes per animal. An increase in the frequency (%) of micro-nucleated polychromatic erythrocytes (%MN-PCEs) in treated animals is an indication of induced chromosome damage. The results obtained for the MN test in rats exposed to CYN/MC-LR mixtures are shown in Table 1, with individual data shown in the Supplementary Materials (Table S1). Significant differences versus the negative and solvent control groups were found in the PCE/total erythrocytes and PCE/NCE ratios in male and female rats treated with the highest assessed dose (75 +750 µ g/kg b.w. CYN/MC-LR; **p<0.01). Treatment with the positive control, ethylmethanesulfonate (EMS), produced similar significant decreases in the PCE/total erythrocytes and PCE/NCE ratios. Furthermore, significant increases in the percentage of MN in immature erythrocytes were observed in all treated groups of both sexes, when compared with the negative and solvent control groups. Toxins 2020,12, 348 4 of 20 Table 1. Micronucleusassaysresults. Bonemarrowcytotoxicityexpressedaspolychromaticerythrocytes (PCE) out of total erythrocytes (normochromatic erythrocytes (NCE) +PCE), ratio of PCE out of NCE, and the micronuclei induction expressed as % MN-PCE ´ s. The values are expressed as mean ± SD. Significantly different from the negative and solvent control (** p<0.01). Groups Sex nDoses PCE/Total % MN-PCE´s PCE/NCE Negative Control (water) ♂5 0.49 ±0.02 0.45 ±0.64 0.95 ±0.08 ♀5 0.49 ±0.03 0.66 ±0.32 0.96 ±0.14 Solvent Control (0.5% MeOH) ♂5 0.50 ±0.02 0.83 ±0.51 0.98 ±0.08 ♀5 0.50 ±0.03 0.58 ±0.24 1.00 ±0.11 Positive Control (EMS *) ♂3200 mg/kg b.w. 0.34 ±0.02 ** 1.93 ±0.15 ** 0.51 ±0.05 ** ♀3 0.36 ±0.03 ** 2.47 ±0.9 ** 0.57 ±0.08 ** CYN/MC-LR ♂5 7.5+75 µ g/kg b.w. 0.51 ±0.02 1.75 ±0.42 ** 1.03 ±0.07 ♀5 0.50 ±0.03 1.98 ±0.21 ** 1.05 ±0.13 ♂5 23.7+237 µ g/kg b.w. 0.46 ±0.04 1.83 ±0.41 ** 0.86 ±0.14 ♀5 0.47 ±0.03 2.09 ±0.15 ** 0.88 ±0.09 ♂5 75+750 µ g/kg b.w. 0.37 ±0.03 ** 1.88 ±0.51 ** 0.60 ±0.08 ** ♀5 0.34 ±0.06 ** 2.22 ±0.27 ** 0.51 ±0.12 ** * EMS: ethylmethanesulfonate. 2.2. Standard and Enzyme-Modified Comet Assay The standard comet assay can detect single and double stranded breaks (SBs), resulting, for example, from direct interactions with DNA, alkali labile sites or as a consequence of transient DNA strand breaks resulting from DNA excision repair [ 54 ]. Moreover, the enzyme-modified comet assay allowed for detection of oxidative DNA damage using the enzymes Endonuclease III (EndoIII) and Formamidopyrimidine DNA glycosylase (Fpg), which detect oxidized pyrimidines and purines, respectively. The results are expressed as the DNA content in the tail (% of DNA in the tail), which is the intensity of the comet tail relative to the total intensity. As positive control, ethylmethanesulfonate (EMS) was chosen. No DNA strand breaks were induced in the standard comet assay at any assessed dose in liver, stomach, and blood cells, with the exception of rats treated with the positive control (EMS) (Figure 1A,B). Furthermore, no increases were observed in the % of DNA in the tail of the comets in liver, stomach and blood cells of both sexes at any exposure assayed after post-treatment with Endo-III (Figure 2). Similarly, no differences were found in the tissues from the treated and control groups after Fpg post-exposure (Figure 3). Significant DNA damage was observed in the studied tissues obtained in all the experiments in the positive control group treated with 200 mg/kg b.w. of EMS, (Figures 1–3). Individual data of alkaline and enzyme-modified comet assays are shown in the Supplementary Materials (Table S1). Summary statistics for the treatment groups (male +female) of both assays are also included in the Supplementary Materials (Table S2). 2.3. Clinical and Histopathological Analysis No clinical signs of toxicity were detected during the experiment at any assayed dose of CYN/MC-LR combinations. No macroscopic changes were observed after necropsy in the gastric mucosa or liver samples of treated groups of both sexes, whereas the stomach had an increased size in the positive control groups. The relative weight (RW) of liver (excised wet liver weight/animal weight) and stomach collected from the treated animals with cyanotoxins was similar to that of the control groups (see Tables S3 and S4 in Supplementary Materials). Toxins 2020,12, 348 5 of 20 Toxins 2020, 12, x FOR PEER REVIEW 5 of 21 Figure 1. The level of DNA damage in cells isolated from liver, stomach, and blood of male (A) and female (B) rats exposed to cylindrospermopsin/microcystins (CYN/MC-LR) mixtures as the formation of strand breaks (SBs) detected by the standard comet assay. The levels of DNA strand breaks are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (***p < 0.001). Figure 2. The level of DNA damage in cells isolated from liver, stomach and blood of male and female rats exposed to CYN/MC-LR combinations as the formation of oxidative DNA damage in the form of Endo-III sensitive sites. The levels of oxidized pyrimidines are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (**p < 0.01). Figure 1. The level of DNA damage in cells isolated from liver, stomach, and blood of male ( A ) and female ( B ) rats exposed to cylindrospermopsin/microcystins (CYN/MC-LR) mixtures as the formation of strand breaks (SBs) detected by the standard comet assay. The levels of DNA strand breaks are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (*** p<0.001). Toxins 2020, 12, x FOR PEER REVIEW 5 of 21 Figure 1. The level of DNA damage in cells isolated from liver, stomach, and blood of male (A) and female (B) rats exposed to cylindrospermopsin/microcystins (CYN/MC-LR) mixtures as the formation of strand breaks (SBs) detected by the standard comet assay. The levels of DNA strand breaks are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (***p < 0.001). Figure 2. The level of DNA damage in cells isolated from liver, stomach and blood of male and female rats exposed to CYN/MC-LR combinations as the formation of oxidative DNA damage in the form of Endo-III sensitive sites. The levels of oxidized pyrimidines are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (**p < 0.01). Figure 2. The level of DNA damage in cells isolated from liver, stomach and blood of male and female rats exposed to CYN/MC-LR combinations as the formation of oxidative DNA damage in the form of Endo-III sensitive sites. The levels of oxidized pyrimidines are expressed as % DNA in the tail of the comets. All values are represented as mean ±SD. Significantly different from control (** p<0.01). The liver and stomach from the negative and solvent control groups showed normal histology without pathological lesions (Figure 4A,B). Degenerate hepatocytes in centrilobular areas together with small, round, and clear vacuoles (microvesicular vacuolation) inside hepatocytes with a diffuse distribution were observed in the liver from rats exposed to medium-lower doses (23.7 +237 and 7.5 +75 µ g/kg b.w. CYN/MC-LR), (Figure 4C,D); however, they did not reach statistical significance with regard to the negative and solvent controls when quantified (Figure 5). Toxins 2020,12, 348 6 of 20 Toxins 2020, 12, x FOR PEER REVIEW 5 of 21 Figure 1. The level of DNA damage in cells isolated from liver, stomach, and blood of male (A) and female (B) rats exposed to cylindrospermopsin/microcystins (CYN/MC-LR) mixtures as the formation of strand breaks (SBs) detected by the standard comet assay. The levels of DNA strand breaks are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (***p < 0.001). Figure 2. The level of DNA damage in cells isolated from liver, stomach and blood of male and female rats exposed to CYN/MC-LR combinations as the formation of oxidative DNA damage in the form of Endo-III sensitive sites. The levels of oxidized pyrimidines are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (**p < 0.01). Figure 3. The level of DNA damage in cells isolated from liver, stomach, and blood of male and female rats exposed to CYN/MC-LR mixtures as the formation of oxidative DNA damage in the form of Formamidopyrimidine glycosilase (Fpg)-sensitives sites. The levels of oxidized purines are expressed as % DNA in the tail of the comets. All values are represented as mean ± SD. Significantly different from control (** p<0.01). Toxins 2020, 12, x FOR PEER REVIEW 7 of 21 Figure 4. Representative histopathological changes in the liver of rats exposed to CYN/MC-LR. Normal hepatic parenchyma is observed in negative (A) and solvent (B) control groups. Details of normal hepatocytes are observed in the insets (A and B). Rats exposed to 7.5 + 75 μg/kg b.w. CYN/MCLR showed a diffuse distribution of mild degenerate hepatocytes with micro-vesicular lipid vacuolation (C). There are details of intracellular accumulation of small, round and clear vacuoles in hepatocytes (C, inset). Rats exposed to 23.7 + 237 μg/kg b.w. CYN/MC-LR presented mild degenerate and necrotic hepatocytes in centrilobular areas (D). There are details of degenerate hepatocytes, some of them multinucleated (*) (D, inset). Rats exposed to 75 + 750 μg/kg b.w. CYN/MC-LR showed moderate degenerate and necrotic hepatocytes in centrilobular areas with macro-vesicular lipid vacuolation (E). There are details of intracellular accumulation of large, round, and clear vacuoles inside hepatocytes, some of them multinucleated (*) (E, inset). The positive control group showed hepatocyte degeneration with lipid accumulation and centrilobular necrosis (F). There are details of the degeneration of hepatocytes with macro-vesicular lipid vacuolation (F, inset). Hematoxylin and eosin staining; bars = 100 μm (A, B, C, D, E) and 50 μm (F). Figure 4. Representative histopathological changes in the liver of rats exposed to CYN/MC-LR. Normal hepatic parenchyma isobservedinnegative( A )andsolvent( B )control groups. Details ofnormalhepatocytes areobservedintheinsets( A , B ).Ratsexposedto7.5+75 µ g/kgb.w. CYN/MC-LRshowedadiffusedistribution of mild degenerate hepatocytes with micro-vesicular lipid vacuolation ( C ). There are details of intracellular accumulation of small, round and clear vacuoles in hepatocytes ( C , inset). Rats exposed to 23.7 +237 µ g/kg b.w. CYN/MC-LR presented mild degenerate and necrotic hepatocytes in centrilobular areas ( D ). There are details of degenerate hepatocytes, some of them multinucleated (*) ( D , inset). Rats exposed to 75 +750 µ g/kg b.w. CYN/MC-LR showed moderate degenerate and necrotic hepatocytes in centrilobular areas with macro-vesicular lipid vacuolation ( E ). There are details of intracellular accumulation of large, round, and clear vacuoles inside hepatocytes, some of them multinucleated (*) ( E , inset). The positive control group showed hepatocyte degeneration with lipid accumulation and centrilobular necrosis ( F ). There are details of the degeneration of hepatocytes with macro-vesicular lipid vacuolation ( F , inset). Hematoxylin and eosin staining; bars =100 µm (A–E) and 50 µm (F). Toxins 2020,12, 348 7 of 20 Toxins 2020, 12, x FOR PEER REVIEW 8 of 21 Figure 5. Mean ± standard error (SE) of the histopathological lesion score from liver and stomach of rats exposed to CYN/MC-LR. Significantly different from control (*p < 0.05; **p < 0.01). Figure 5. Mean ± standard error (SE) of the histopathological lesion score from liver and stomach of rats exposed to CYN/MC-LR. Significantly different from control (* p<0.05; ** p<0.01). Similar, but more severe liver lesions than in the lower doses were observed in the highest exposed group (75 +750 µ g/kg b.w. CYN/MC-LR). In this case, degenerate and necrotic hepatocytes in centrilobular areas were observed, as well as ample intracellular lipid accumulation in hepatocytes in the form of round and clear vacuoles (macrovesicular vacuolation). These had a multifocal distribution together with mild presence of multinuclear hepatocytes (Figure 4E). This group showed significant liver damage compared to negative and solvent control groups (Figure 5). The liver pathology of the positive controls was characterized by severe centrilobular necrosis, hepatocyte degeneration with lipid accumulation, and mild mononuclear inflammatory periportal infiltrates (Figure 4F). This group showed significant differences in the lesion scores compared to negative and solvent control groups (Figure 5). On the other hand, the main damage in the stomach included flaking epithelium and minimal-mild necrosis of epithelial cells with different severity, depending on the groups. These lesions were moderate in the positive controls accompanied by hyperplasia of the gastric glands, while being mild in the highest exposed group, minimal in the medium-lower doses, and totally absent in negative and solvent controls (Figure 6A–F). Statistical differences were observed only between positive control and the rest of the studied groups (Figure 5). Toxins 2020,12, 348 8 of 20 Toxins 2020, 12, x FOR PEER REVIEW 9 of 21 Figure 6. Representative histopathological changes in the stomach of rats exposed to CYN/MC-LR. Normal gastric mucosa is observed in negative (A) and solvent (B) control groups. Rats exposed to 7.5 + 75 and 23.7 + 237 μg/kg b.w. CYN/MC-LR showed minimal flaking gastric epithelium (arrows) (C and D, respectively). Rats exposed to 75 + 750 μg/kg b.w. CYN/MC-LR showed a mild flaking gastric epithelium with occasional necrosis of epithelial cells and a mild increase of the secretory epithelium (arrow) (E). Positive control group presented a moderate flaking gastric epithelium with mild necrosis of epithelial cells and hyperplasia of the gastric glands (arrows) (F). Hematoxylin and eosin staining; bars = 100 μm (A, C, D), 75 μm (B, E) and 50 μm (F). The lesions were independently examined by two experienced assessors: a veterinary pathologist and an investigator (M.A.R. and R.M.) in a single-blinded approach. These findings were evaluated in all the fields of one slide with a section of 1 × 1 cm from each organ studied in all the animals 3. Discussion Although the simultaneous occurrence of cyanotoxins such as MCs and CYN is being reported more and more frequently [44,55], toxicological studies focusing on their potential interaction are very scarce [50,56]. Particularly, the genotoxicity of mixtures is of great interest for humans exposed to contaminated waters and food with cyanotoxins, due to the possible carcinogenic effects of MCLR and pro-genotoxic activity of CYN. Moreover, as EFSA recommends [47], further studies are needed to characterize the hazard for a more realistic and reliable risk assessment. Only two in vitro studies evaluated the genotoxic effects of CYN/MC-LR mixtures. The first was carried out on the HepG2 cell line exposed to MC-LR (1 μg/mL), CYN (0.01, 0.05, 0.1, and 0.5 μg/mL) and their mixtures by comet and MN assay [48]. The authors indicated that CYN might have a higher Figure 6. Representative histopathological changes in the stomach of rats exposed to CYN/MC-LR. Normal gastric mucosa is observed in negative ( A ) and solvent ( B ) control groups. Rats exposed to 7.5 +75 and 23.7 +237 µ g/kg b.w. CYN/MC-LR showed minimal flaking gastric epithelium (arrows) ( C , D , respectively). Rats exposed to 75 +750 µ g/kg b.w. CYN/MC-LR showed a mild flaking gastric epithelium with occasional necrosis of epithelial cells and a mild increase of the secretory epithelium (arrow) ( E ). Positive control group presented a moderate flaking gastric epithelium with mild necrosis of epithelial cells and hyperplasia of the gastric glands (arrows) ( F ). Hematoxylin and eosin staining; bars =100 µ m ( A , C , D ), 75 µ m ( B , E ) and 50 µ m ( F ). The lesions were independently examined by two experienced assessors: a veterinary pathologist and an investigator (M.A.R. and R.M.) in a single-blinded approach. These findings were evaluated in all the fields of one slide with a section of 1 ×1 cm from each organ studied in all the animals. 3. Discussion Although the simultaneous occurrence of cyanotoxins such as MCs and CYN is being reported more and more frequently [ 44 , 55 ], toxicological studies focusing on their potential interaction are very scarce [ 50 , 56 ]. Particularly, the genotoxicity of mixtures is of great interest for humans exposed to contaminated waters and food with cyanotoxins, due to the possible carcinogenic effects of MC-LR and pro-genotoxic activity of CYN. Moreover, as EFSA recommends [ 47 ], further studies are needed to characterize the hazard for a more realistic and reliable risk assessment. Only two in vitro studies evaluated the genotoxic effects of CYN/MC-LR mixtures. The first was carried out on the HepG2 cell line exposed to MC-LR (1 µ g/mL), CYN (0.01, 0.05, 0.1, and 0.5 µ g/mL) and their mixtures by comet and MN assay [ 48 ]. The authors indicated that CYN might have a higher genotoxic potential than MC-LR and the genotoxic potential of CYN/MC-LR combination was similar to CYN alone. More recently, the mutagenicity and genotoxicity of 1:10 CYN/MC-LR mixtures were assayed through a complete battery of in vitro tests including the MN test on L5178Y Tk ± cells and the Toxins 2020,12, 348 9 of 20 standard and enzyme-modified comet assays in Caco-2 cells [ 49 ]. Genotoxicity was observed only for the combination in the MN test with S9 metabolic fraction, in agreement with the previous reports for only CYN [ 57 ]. Together, both in vitro studies suggested the predominance of the pro-genotoxic activity of CYN in the combinations, hence the necessity to evaluate in vivo the genotoxicity of CYN/MC-LR mixtures by application of international guidelines such as the OECD guidelines. This is the first in vivo study which reported the genotoxicity of CYN/MC-LR in rats orally exposed (by gavage). It combines and relates the results of the two types of comet assays (standard and enzyme-modified) on cells isolated from stomach, liver, and blood, with the MN test in bone marrow cells. The assays were performed according to the OECD 489 and 474 guidelines, respectively [ 53 , 54 ] with some alterations, as described by Diez-Quijada et al. [ 43 ]. The combined comet-MN assay [ 58 ] reduces the use of animals according to the 3Rs principles (Replace, Reduce, and Refine), and it increases the sensitivity and specificity of the assays, decreasing the number of false negative results [ 59 ]. Moreover, in this case, the use of DNA repair enzymes (Endo III and Fpg) was also included, increasing the sensitivity of the in vivo comet assay [60,61]. In this study, the combined MN-comet assay was performed in both sexes, because differences in toxicity linked to gender have previously been described. This is in agreement with the OECD 489 [ 54 ] that encourages the use of both sexes when relevant differences between males and females are reported (e.g. differences in systemic toxicity, metabolism, bioavailability, etc. including those in a range-finding study). In this sense, although no sex-related differences were observed in rats after MC-LR i.p. exposure [ 62 ], male mice were more sensitive than females to CYN administered i.p. [ 63 ]. CYN exposure doses (75, 23.7, and 7.5 µ g/kg b.w.) were selected according to a previous in vivo study performed with the individual toxin [ 43 ]. The doses of MC-LR were 10 times higher than CYN because of its proportionally higher abundance in the environment [ 64 ]. Overall in this study, using rats as experimental model, the CYN/MC-LR combinations increased the % MN-PCE in bone marrow cells, although it induced no DNA strand breaks nor any oxidative DNA damage in any of the investigated tissues. These results are in accordance with the only two previous genotoxicity in vivo assay carried out in rats with pure individual toxins: CYN alone under the same combined assays [ 43 ] and MC-LR administered i.v. through the comet assay [ 28 ]. Generally, the results of this study show no synergism or antagonism between CYN and MC-LR in the assayed combinations and concentrations in rats, because the effects are similar to the in vivo exposure to CYN alone [ 43 ]. Moreover, these findings confirm the in vitro experiments performed with the CYN/MC-LR mixture through a battery of tests, in which MN and comet assays were included [49]. In the present study, the significant decreases in the PCE/total erythrocytes and PCE/NCE ratios in the highest dose groups (75 +750 µ g/kg b.w. CYN/MC-LR) in comparison to the controls were consistent with results observed for CYN tested individually at the same doses (7.5–75.0 µ g/kg b.w.) [ 43 ]. Previously, in the case of MC-LR no in vivo effects were reported for polychromatic erythrocytes (PCE) from peripheral blood of male mice after i.p. administration of the toxin (0–55 µ g/kg b.w.) nor induction of MN [19]. Moreover, the induction of MN by the CYN/MC-LR mixture in rats contrasts with the contradictory results obtained in previous assays performed in mice as experimental model. Thus, negative results were reported in the colonic cells of mice orally exposed (1–4 mg/kg b.w.) and in bone marrow after i.v. administration [ 41 ]. In contrast, positive results were found in the blood cells of mice exposed i.p. to 37.5 µ g/kg b.w. MC-LR, with significant increases in the frequency of MN at 48–72 h after treatment [ 26 ]. Additionally, only a rapid and temporary two-fold increase had been detected in the amounts of DNA in the tail of the comets after 30 min of MC-LR exposure was detected in the comet assay. The authors suggested that although MC-LR induced DNA damage, the leukocytes might repair the lesions, prior to the genotoxicity assessment by the comet assay. They concluded that the MN assay could be more sensitive than the comet assay to evaluate the genotoxicity of MC-LR. This hypothesis could also explain the results obtained in the present work. Other studies showed a significant dose-dependent increase in MN frequency of bone marrow cells of Balb/C mice i.p. injected with MC-LR (0.5–8 µ g/kg Toxins 2020,12, 348 16 of 20 17. Puerto, M.; Pichardo, S.; Jos, Á .; Prieto, A.I.; Sevilla, E.; Fr í as, J.E.; Came á n, A.M. 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