Effect concentrations in the toxicity assessment of chemicals with fish and fish cells are generally based on external exposure concentrations. External concentrations as dose metrics, may, however, hamper interpretation and extrapolation of toxicological effects because it is the internal concentration that gives rise to the biological effective dose. Thus, we need to understand the relationship between the external and internal concentrations of chemicals. The objectives of this study were to: (i) elucidate the time-course of the concentration of chemicals with a wide range of physicochemical properties in the compartments of an in vitro test system, (ii) derive a predictive model for toxicokinetics in the in vitro test system, (iii) test the hypothesis that internal effect concentrations in fish (in vivo) and fish cell lines (in vitro) correlate, and (iv) develop a quantitative in vitro to in vivo toxicity extrapolation method for fish acute toxicity. To achieve these goals, time-dependent amounts of organic chemicals were measured in medium, cells (RTgill-W1) and the plastic of exposure wells. Then, the relation between uptake, elimination rate constants, and log KOW was investigated for cells in order to develop a toxicokinetic model. This model was used to predict internal effect concentrations in cells, which were compared with internal effect concentrations in fish gills predicted by a Physiologically Based Toxicokinetic model. Our model could predict concentrations of non-volatile organic chemicals with log KOW between 0.5 and 7 in cells. The correlation of the log ratio of internal effect concentrations in fish gills and the fish gill cell line with the log KOW was significant (r>0.85, p = 0.0008, F-test). This ratio can be predicted from the log KOW of the chemical (77% of variance explained), comprising a promising model to predict lethal effects on fish based on in vitro data.

Permanent fish cell lines constitute a promising complement or substitute for fish in the environmental risk assessment of chemicals. We demonstrate the potential of a set of cell lines originating from rainbow trout ( Oncorhynchus mykiss) to aid in the prediction of chemical bioaccumulation in fish, using benzo[ a]pyrene (BaP) as a model chemical. We selected three cell lines from different tissues to more fully account for whole-body biotransformation in vivo: the RTL-W1 cell line, representing the liver as major site of biotransformation, and the RTgill-W1 (gill) and RTgutGC (intestine) cell lines, as important environment-organism interfaces, which likely influence chemical uptake. All three cell lines were found to effectively biotransform BaP. However, rates of in vitro clearance differed, with the RTL-W1 cell line being most efficient, followed by RTgutGC. Co-exposures with α-naphthoflavone as potent inhibitor of biotransformation, assessment of CYP1A catalytic activity, and the progression of cellular toxicity upon prolonged BaP exposure revealed that BaP is handled differently in the RTgill-W1 compared to the other two cell lines. Application of the cell-line-derived in vitro clearance rates into a physiology-based toxicokinetic model predicted a BaP bioconcentration factor (BCF) of 909-1057 compared to 920 reported for rainbow trout in vivo.

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine's anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain.

Cells in vitro can save lives of experimental fish used in chemical safety testing.

No abstract

The acceptance and usefulness of simulation models are often limited by the efficiency, transparency, reproducibility, and reliability of the modelling process. We address these issues by suggesting that modellers (1) "trace" the iterative modelling process by keeping a modelling notebook corresponding to the laboratory notebooks used by empirical researchers, (2) use a standardized notebook structure and terminology based on the existing TRACE documentation framework, and (3) use their notebooks to compile TRACE documents that supplement publications and reports. These practices have benefits for model developers, users, and stakeholders: improved and efficient model design, analysis, testing, and application; increased model acceptance and reuse; and replicability and reproducibility of the model and the simulation experiments. Using TRACE terminology and structure in modelling notebooks facilitates production of TRACE documents. We explain the

The maximal chemical concentration that causes an acceptably small or no effect in an organism or isolated cells is an often-sought-after value in toxicology. Existing approaches to derive this value have raised several concerns; thus, it is often chosen case-by-case based on personal experience. To overcome this ambiguity, we propose an approach for choosing the non-toxic concentration (NtC) of a chemical in a rational, tractable way. We developed an algorithm that identifies the highest chemical concentration that causes no more than 10% effect (= EC10) including the modeled 95% confidence intervals and considering each of the measured biological replicates; and whose toxicity is not significantly different from no effect. The developed algorithm was validated in two steps: by comparing its results with measured and modeled data for 91 dose-response experiments with fish cell lines and/or zebrafish embryos; and by measuring actual effects caused by NtCs in a separate set of experiments using a fish cell line and zebrafish embryos. The algorithm provided an NtC that is more protective than NOEC (no-observed-effect-concentration), NEC (modeled no-effect concentration), EC10 and BMD (benchmark dose). Despite focusing on small-scale bioassays here, this study indicates that the NtC algorithm could be used in various systems. Its application to the survival of zebrafish embryos and to metabolic activity in cell lines showed that NtCs can be applied to different effect measurements, time points, and levels of biological organization. The algorithm is available as Matlab and R source code, and as a free, user-friendly online application.

The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the ever-increasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish (Danio rerio) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration.