Objective Intro Materials Procedure Results 1 Results 2 Results 3 Discussion Lit. Cited Prep Sheet


Arsenic, a metal pollutant found naturally in groundwater and unnaturally in mine waste sites and agricultural runoff, has been considered toxic to humans for several millennia and has recently been discovered to be a human carcinogen as well (Shalat et al., 1996). Arsenic has similar toxic effects on a range of organisms, with effects on mature organisms ranging from cancers and nerve damage in humans (Shalat et al., 1996) to metabolic changes and learning deficiencies in rats (Rodríguez et al., 2002). Many studies have also examined arsenic’s effect on embryological development to determine the signaling pathways that the toxin impacts. Studies have suggested that arsenic is teratogenic, and it has been shown to cross the mammalian placenta, affecting developing embryos whose mothers undergo exposure (Wlodarczyk et al.,1996).

Arsenic exists in both trivalent and pentavalent ionic forms, and is often given as either sodium arsenate (As(V)) or arsenic trioxide (As(III)), with sometimes opposing results. Injections of arsenic trioxide to pregnant rats caused embryonic defects when the dosage was great enough to cause severe adverse effects on the mother, whereas comparable amounts of sodium arsenate impacted the mothers less acutely while causing higher incidences of more severe defects in the embryos (Stump et al., 1999). Contrarily, Peterková and Puzanová (1974) showed that As(V) was less toxic to chick embryos than As(III). The dosages of both directly correlated to their toxicity in this experiment (Peterková and Puzanová 1974); but in rainbow trout (Salmo gairdneri), embryo survival increased with pentavalent arsenic concentration while trivalent arsenic concentrations had no clear relationship with mortality rates (Dabrowski 1975). The trout embryos had lower survival in pentavalent arsenic than in control solutions, but among arsenic solutions the higher concentrations exhibited lower mortality than lower concentrations. Some studies have suggested that As(V) is less toxic to fish than As(III), an identical result to findings in higher vertebrates (Dabrowski 1975), although the effects on rat embryos described above oppose this (Stump et al., 1999). Once again in opposition to results anticipated based upon previous findings in fish, direct correlation was seen between concentration and mortality rates when Japanese medaka (Oryzias latipes) embryos were exposed to arsenic trioxide (Tchounwou et al., 2001). Only the trivalent ion of arsenic has direct biological ramifications: As(V) must be converted to As(III) to be active (Peterková and Puzanová 1974), so the greater influence of pentavalent arsenic in some cases is surprising. Kalter (1968) noted that the effect of teratogens in organisms such as fish and amphibians is primarily dependent upon the stage at which the embryo is exposed to the chemical, offering a potential explanation for the discrepant results outlined above. Arsenic has also been found to select organs on which to act based on whether the critical period of their development coincided with the stage at which it is administered (Peterková and Puzanová 1974). However, response to arsenic is also species dependent and therefore cannot be generalized based on broader classifications.

Aside from its impact on mortality rates, arsenic has been found to cause miscarriage, stillbirth, developmental retardation and birth defects in humans (Aaschengrau et al., 1989 for example, as cited by Shalat et al., 1996); neural tube, ocular and jaw defects (Stump et al., 1999) and behavioral retardation and delayed ear detachment (Rodriguez et al., 2002) in rodent embryos; and everted internal organs and small eyes in chicks (Gilani and Alibhai 1990, as cited by Shalat et al., 1996). Neural tube defects have been identified as a common defect among mammals, possibly reflecting the fact that arsenic accumulates in the neuroepithelium of developing fetuses (Shalat et al., 1996). Inorganic arsenic, of which sodium arsenate and arsenic trioxide are two kinds, may cause neural tube defects by repressing cell replication through microtubule organization or by inhibiting cell shape changes necessary to neural tube formation (Shalat et al., 1996). Wlodarczyk and coworkers (1996) suggest that arsenic damaging of DNA is responsible for inhibition of cell propagation, thus delaying and preventing a normal neural tube closure.

This experiment examines the effect of inorganic pentavalent arsenic, administered in the form of sodium arsenate, on developing zebrafish (Danio rerio) embryos. The effects of arsenic on fish have environmental ramifications as arsenic is present in some water systems throughout the world and accumulates in aquatic animals (Tchounwou et al., 2001), sometimes to concentrations that would be toxic to humans (Chapman 1926, as cited by Dabrowski 1975).

Zebrafish make good test subjects as embryos can be easily harvested year round and at selected times of day. Their advantage over other fish species lies in their relatively rapid development, and their optically clear embryos. Arsenic’s impact on fish embryos may vary greatly from its impact on embryos from other classes because fish undergo a different process of neurulation from many other vertebrates. Their neural tube does not close over to form a tube but rather hollows from a solid core of cells. Thus, neural tube defects may manifest themselves differently. While the effects of arsenic on rainbow trout (Salmo gairdneri) (Dabrowski 1975) and Japanese medaka (Oryzias latipes) (Tchounwou et al., 2001) embryos have been investigated, no studies of zebrafish have been performed. As arsenic influence varies so dramatically between species, including the two fish species mentioned, an investigation into the effects of arsenic on zebrafish merits assay. Additionally, the two studies on fish looked at toxicity and accumulation but not morphological defects, making this investigation unique.

In this investigation, zebrafish (Danio rerio) embryos will be exposed to various concentrations of arsenic(V) at five, eight and twelve hours after fertilization to observe the effects of this teratogen on neural tube development. At five hours, most embryos are at approximately 50% epiboly. At this stage, the embryonic shield is just forming, so cells have not yet differentiated (Kimmel et al., 1995). At eight hours, embryos are between 60 and 100% epiboly and the axes begin to form as cells converge to create the basic form of the embryo curled around the yolk sac (Kimmel et al., 1995). This stage was chosen because neurulation succeeds it closely. At twelve hours, neurulation is underway, with neural keel formation about to take place (Kimmel et al., 1995).

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