Potential of the zebrafish (Danio rerio) embryo test to discriminate between chemicals of similar molecular structure—a study with valproic acid and 14 of its analogues

Liver toxicity in the zebrafish embryo

For the correct identification of adverse liver effects in species from different vertebrate classes, three parameters have to be considered: (1) similarities and differences of hepatogenesis, anatomy and morphology of the zebrafish liver relative to mammals, (2) the rapid development of the zebrafish embryo within the first days of its life and, hence, (3) a different morphological state of the liver at each developmental stage. Although knowledge about the morphology and ultrastructure of adult Danio rerio liver, which has been described in detail by Braunbeck et al. (1990), Menke et al. (2011), as well as Yao et al. (2012), may contribute to understand the general anatomy and functionality of the liver in fish, additional information is required as a basis for the toxicological evaluation of embryonic hepatocytes.

In mice, hepatogenesis starts at approximately one-third of the way through gestation and is only completed near birth (Chu and Sadler 2009). It starts with the establishment of a population of hepatic precursor cells within the ventral foregut endoderm, which specify into definite hepatoblasts (liver progenitor cells). These precursor cells delaminate from the epithelial layer to form a liver bud, proliferate rapidly and finally differentiate into functional hepatocytes and biliary duct cells (Tao and Peng 2009).

In zebrafish, hepatogenesis is divided into three stages: specification, differentiation and hepatic outgrowth (Chu and Sadler 2009; Hill 2012; Tao and Peng 2009; Wilkins and Pack 2013). During specification, liver progenitor cells originating from the anterior endoderm are identifiable earliest at 16 h and latest between 22 and 24 h by the expression of hhex and prox-1 (Chu and Sadler 2009; Tao and Peng 2009; Wilkins and Pack 2013). Among endoderm-derived organs (i.e., intestine, pancreas, hepatopancreatic ductal system or pneumatic duct and swim bladder), the liver is the organ determined first (Wilkins and Pack 2013). This observation underlines its developmental and evolutionary relevance for the zebrafish embryo, since growth of the whole organism is linked to the exploitation of yolk through the liver.

In the second stage, hepatoblasts aggregate between 24 and 28 h, which leads to the thickening in the intestinal primordium (Hill 2012), and the initiation of differentiation. Molecular markers of mature hepatocytes and biliary epithelial cells are detectable at 32 h (ceruloplasmin) and 48 h (transferrin and L-FABP) (Chu and Sadler 2009; Wilkins and Pack 2013), and CYP-mediated metabolism (be it hepatocellular or extrahepatic) is already active at 36 h (Lörracher and Braunbeck 2020). At 48 h, the liver primordium is clearly discernable as a prominent bud extending from the left of the midline over the yolk (Chu and Sadler 2009), and at approximately 50 h liver tissue is easily recognizable (Hill 2012). At the end of this stage, the liver is located anteriorly between the duct of Cuvier and posteriorly the mid-level of the fin bud (Tao and Peng 2009).

Finally, in the third stage of zebrafish development, the liver changes in size, shape and localization by a rapid proliferation, differentiation and polarization of hepatocytes and the expansion of the biliary system (Chu and Sadler 2009; Wilkins and Pack 2013). This growth phase is initiated approximately at 50 h and continues into the juvenile stage, until the liver parenchyma and the biliary tract are fully developed (Chu and Sadler 2009; Wilkins and Pack 2013).

Between 55 and 72 h, growth of the hepatic vasculature is initiated to facilitate the rapid growth of the organ. Endothelial cells partially encapsulate the liver bud and subsequently start to invade it (Chu and Sadler 2009; Wilkins and Pack 2013). By 72 h, “vascularization is essentially completed, and the liver becomes perfused with blood shortly after” (Hill 2012). At 96 h, the zebrafish liver consists of a larger left lobe that crosses the midline ventral to the esophagus, and forms the smaller right lobe that extends ventrally towards the head of the pancreas. It touches the pericardial cavity anteriorly and overlaps with the anterior portion of residual yolk (Chu and Sadler 2009; Field et al. 2003; Tao and Peng 2009).

At 5 days post fertilization, zebrafish liver embryogenesis is essentially complete, the digestive system is basically functional (Hill 2012), and bile production, serum protein secretion, glycogen storage and lipogenesis are fully operational (Chu and Sadler 2009).

In principle, the developmental stages described in the zebrafish embryo match with hepatogenesis in mammals; however, there are four major differences: (1) For mammalian liver development, hepatic vasculature and hematopoiesis are essential. Mutations of these systems often cause anemia and early lethality, which might lead to complications in the study of liver development (Tao and Peng 2009). This is not the case with zebrafish, since embryonic hematopoiesis does not take place in the zebrafish liver. In fact, zebrafish early liver development is independent of vasculogenesis, which allows the embryo to develop for several days even without cardiovascular circulation (Korzh et al. 2008; Tao and Peng 2009). (2) During the development and differentiation of the hepatic bud in mammals, the septum transversum mesenchyme provides important inductive signals. This structure does not seem to exist in fish; however, the lateral plate mesoderm apparently has an analogous function in zebrafish (Chu and Sadler 2009). (3) The cellular and histological architecture clearly differ between mammals and zebrafish, although these still seem to maintain the same functions, which have already been studied in medaka (Oryzias latipes) (Hardman et al. 2007): whereas mammalian livers regularly show portal triads consisting of an artery, a larger vein and a bile duct, teleost fish hepatocytes are more typically organized in plates (hepatocellular cords) lined by sinusoids and biliary ductules, as ramifications of a more irregular biliary tract (Chu and Sadler 2009). (4) In mammals, the biliary system itself consists of extra- and intrahepatic ducts and ductules, whereas in fish preductal epithelial cells are an extra branch on the teleost biliary tree and analogues to the Canal of Hering, which form junctions with canaliculi to collect the bile (Chu and Sadler 2009; Hardman et al. 2007). According to Chu and Sadler (2009), these cells might represent the fish version to hepatic progenitors in other organisms.

Despite these differences, the final general anatomy, organization, cellular composition and function of a healthy adult zebrafish liver are virtually the same as in mammals, and the early embryonic stages of hepatogenesis are similar to that of mice (Hill 2012). Drug metabolization operates similar to human, their metabolic reactions include oxidation, hydroxylation, conjugation, demethylation and deethylation (Lörracher and Braunbeck 2021; Vliegenthart et al. 2014). Likewise, with regard to disease phenotypes, the histopathological syndromes of cholestasis, fatty liver (steatosis) and neoplasia as well as liver regeneration and hepatocarcinogenesis also appear principally comparable in both organisms, even in 5 d old larvae (Amali et al. 2006; Goessling and Sadler 2015; Hill 2012; Spitsbergen et al. 2000), although the processes leading to the phenotypes might be different in detail. Furthermore, extensive research into genetics and tissue cultures uncovered a network of transcription factors and signaling pathways, which are required for forming not only the mammalian liver, but are essential for zebrafish hepatogenesis as well (Chu and Sadler 2009; Tao and Peng 2009; Wilkins and Pack 2013).

Based on the knowledge of these developmental stages, morphology of histological liver sections of non-treated and solvent control embryos becomes reasonable: liver sections stretching from a big left lobe to a smaller right lobe, displaying blood vessels filled with blood cells, as well as multiple regularly shaped nuclei, indicate the proliferation and outgrowth process of a healthy organ at the end of stage three of hepatogenesis in zebrafish (Fig. 1a, b) (Chu and Sadler 2009; Wilkins and Pack 2013).

In contrast, observations made in treated zebrafish embryos indicate numerous symptoms of liver alteration. In the present study, the reduced diameter of hepatocytes was considered as the most important endpoint for the evaluation of histological changes after exposure to valproic acid and its analogues. Other observations included a conspicuous reduction of storage materials, reduced or even missing vascularization (i.e., no or only erratic blood cells between hepatocytes as well as irregular nuclei). The apparent concentration-dependent functional restriction of the liver cells finds its correlate in an overall decline of liver size; this endpoint, however, was ascribed least importance, since the size of an organ can also be linked to the overall size (developmental stage) of the embryo, which has unfortunately not been measured in detail in the present study and could, therefore, not be investigated further (Fig. 1c). Taken together, liver effects recorded in embryos exposed to VPA and its analogues suggest a morphology typical of the end of hepatogenesis stage two. This conclusion could be drawn for all compounds tested within Groups 1 and 2, except for hexanoic acid and 4-pentenoic acid.

There are, however, also controversial observations in previous studies by Passeri et al. (2009), Thakur et al. (2011), as well as Driessen et al. (2013), who described a hepatocellular structure in negative control zebrafish embryos similar to that seen in embryos treated with VPA or its analogues in the present study. This discrepancy is likely due to differences in the fixation procedures: Whereas Passeri et al. (2009), Thakur et al. (2011), as well as Driessen et al. (2013) used only 4% (v/v) paraformaldehyde as fixatives, the present study used a more complex mixture, Davidsons’s fluid, which is known to cause less tissue shrinkage and distortion (Lang 2006; Leimbacher 2009; Simmons and Swanson 2009; Small and Peterson 1982).

Other endpoints such as, e.g., accumulation of vesicular lipid deposits, which might indicate steatosis and were also described by Passeri et al. (2009) and Driessen et al. (2013), could not be confirmed in the present study, since, for an unequivocal evaluation, another fixation and staining method would have been required, namely LipidGreen 2 staining (Chun et al. 2013) or 4% (v/v) paraformaldehyde fixation, followed by PAS-Alcian blue staining (Mulisch and Welsch 2015).

Overall, the liver alterations observed for VPA and 12 of 14 analogues might either be interpreted as a hepatotoxic effect, a retardation or partial inhibition of liver developmental (Cox and Goessling 2015; Farooq et al. 2008). The latter could also be induced indirectly by side effects and might be reversible after termination of the treatment (Raldua et al. 2008). The lack of information about the initiating molecular effects by VPA or its analogues complicates the identification of plausible causes. However, epigenetic experiments revealed that histone deacetylase (HDAC) or DNA methyltransferase activities control both hepatic specification and outgrowth (Chu and Sadler 2009; Farooq et al. 2008). Treating zebrafish embryos with an HDAC inhibitor prior to 24 h reduces hhex and prox-1 expression, resulting in a smaller liver (Chu and Sadler 2009). In specific, hdac1 and hdac 3 seem to be involved in patterning and hepatic outgrowth (Chu and Sadler 2009), and, since VPA has been shown to be an HDAC inhibitor in both mammals and zebrafish (Giavini and Menegola 2014; Gurvich et al. 2005; Li et al. 2016; Massa et al. 2005), the liver alterations described might in fact be liver-specific effects and not secondary teratogenic effects.

Moreover, apart from genetic and epigenetic alterations, the pH shift into a slightly acidic milieu can also not be excluded as a trigger for the effects observed. Although there are no reports in literature on pH-dependent changes in zebrafish liver architecture and zebrafish embryos are regarded to be fairly tolerant to pH variations between pH 6.5 and 8.5 (OECD 2013), it should still be noted that pH may profoundly affect the specification and solubility of the test solutions, thus changing the availability of the compounds to zebrafish embryos.

Discrimination of molecular similarity of analogues by a structure–activity relationship

Although the histopathological observations can per se neither categorize a definite in vivo-positive or negative potency for liver toxicity nor identify steatosis, calculations and subsequent analyses of EC20 values for liver-altering effects clearly allowed to correlate a decrease of hepatotoxic activity with decreasing side chain length.

This observation corroborates similar conclusions by Herrmann (1993); however, the length of side chains is not the sole determinant for the hepatotoxic potency, since the number of side chains apparently also plays an important role. Compounds with one side chain were non-toxic; substances with three side chains, namely two short and one long side chain, were less toxic than those with two side chains. Furthermore, symmetry of the side chains seemed to be another important parameter, as was evident for, e.g., VPA and 2-ethylhexanoic acid: Both compounds have the same number of carbon atoms; however, while both side chains of VPA are equally long, 2-ethylhexanoic carries a longer and shorter side chain, which decreases its toxicity potential. This rule could be confirmed for the comparison of 2-ethylbutyric acid and 2-methylpentanoic acid (Table 4) and was also observed in two human cell lines, namely HepG2 and HepaRG (Escher et al. 2022). Finally, compounds with higher KOW had lower EC20 values and showed higher hepatotoxic potencies (Table 4); hence, compounds with high lipophilicity seem to be better accessible by the zebrafish embryo, which can also lead to elevated accumulation within the organism (de Koning et al. 2015). An additional parameter influencing the absorption of substances is pH-dependency of acids such as those tested in the present study. Although zebrafish embryos are quite tolerant to pH variations between pH 6.5 and 8.5 (OECD 2013), pH may certainly affect both speciation and solubility of the test compounds by manipulating the ratios between ionized and non-ionized molecules and, thus, manipulating the availability of the compounds to the embryos. In case of pH adjustment, the observed SAR-trend would become more distinguished due to differential absorption capacities and low activity analogues would even have needed relatively higher (nominal) test concentrations for inducing hepatotoxic effects at all, thus confirming the current conclusions. For confirmation, a comparison of bioavailability in pH-adjusted versus non-adjusted test scenarios is underway.

As a consequence, results suggested a high potency for liver-altering effects for dicarboxylic acids with long side chains, namely 2-n-propylheptanoic acid, valproic acid, 2-ethylhexanoic acid and 4-ene valproic acid. In contrast, monocarboxylic acids (hexanoic acid and 4-pentenoic acid) did not show any alterations even at the highest concentrations tested. Interestingly, three out of four chemicals expressing the highest potency liver alteration in the FET (valproic acid, 2-ethylhexanoic acid, 4-ene valproic acid) were also in vivo-positive in mice and rats with respect to the development of steatosis (Abdel-Dayem et al. 2014; BG Chemie 2000; Espandiari et al. 2008; Ibrahim 2012; Juberg et al. 1998; Kassahun and Abbott 1993; Knapp et al. 2008; Löscher et al. 1992; Patel and Sanyal 2013; Sugimoto et al. 1987; Tang et al. 1995; Tong et al. 2005; Willebrords et al.

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