Methods to detect non-genotoxic carcinogens using flatworms
10197556 ยท 2019-02-05
Assignee
Inventors
- Karen Smeets (Bilzen, BE)
- An-Sofie Stevens (Maaseik, BE)
- Michelle Plusquin (Bilzen, BE)
- Tom Artois (Diepenbeek, BE)
- Maxime Willems (Balegem, BE)
- Jean Paul Remon (Melle, BE)
Cpc classification
G01N2333/47
PHYSICS
International classification
G01N31/00
PHYSICS
G01N33/53
PHYSICS
Abstract
This disclosure relates to predicting the carcinogenicity of compounds. More in particular, this application discloses methods to detect whether a compound is a non-genotoxic carcinogen and methods to discriminate between genotoxic and non-genotoxic carcinogens based on in vivo stem cell proliferation patterns in flatworms.
Claims
1. A method for determining whether a compound is a genotoxic carcinogen, the method comprising: cutting a flatworm transversally in order to create at least one regenerating flatworm, exposing a regenerating flatworm therefrom to the compound for a period of time and at a concentration that triggers phenotypic effects in regenerating flatworms, determining the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, within said regenerating flatworm after exposure to the compound, wherein the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells is determined with a phosphorylated histone H3 immunoassay, and comparing the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, within the regenerating flatworm after exposure to the compound to the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, within a regenerating flatworm obtained by a similar exposure to a non-carcinogenic, control compound, so as to classify the compound as a genotoxic carcinogen when the determined number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, is significantly lower than the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, within a regenerating flatworm after the similar exposure to the non-carcinogenic, control compound.
2. The method according to claim 1, wherein the period of time that triggers phenotypic effects in regenerating flatworms is between 3 and 19 days.
3. The method according to claim 1, wherein said phenotypic effects are selected from the group consisting of aberrant pigmentation spots, tissue regression, lesions, blisters, bloating, behavioral changes, difficulties in regeneration, abnormal body size, tissue outgrowth and flattened posture.
4. A method of detecting whether a compound is a non-genotoxic carcinogen, the method comprising: cutting a flatworm transversally in order to create at least one regenerating flatworm, exposing the regenerating flatworm to the compound for at least three (3) days, determining the number of adult pluripotent stem cells, execluding gonadal phosphorylated H3-positive cells, within said regenerating flatworm after said exposure to the compound, wherein the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, is determined with a phosphorylated histone H3 immunoassay, and comparing the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, within the regenerating flatworm after exposure to the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, within a regenerating flatworm obtained by a similar exposure to a non-carcinogenic, control compound, so as to classify the compound, wherein the compound is classified as a non-genotoxic carcinogenic when there is a concentration-dependent increase in the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, at day 3 post-exposure, wherein the compound is classified as a genotoxic carcinogenic when there is a concentration-dependent decrease in the number of adult pluripotent stem cells, excluding gonadal phosphorylate H3-positive cells, at day 3 post-exposure, and wherein the compound is classified as non-carcinogenic when there is no concentration-dependent change in the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, at day 3 post-exposure.
5. The method according to claim 1, wherein the flatworm is Macrostomum lignano or Schmidtea mediterranea.
6. The method according to claim 4, wherein the flatworm is Macrostomum lignano or Schmidtea editerranea.
7. A method for determining whether a compound is a genotoxic carcinogen, the method comprising: cutting a flatworm transversallv in order to create at least one regenerating flatworm, exposing a first regenerating flatworm to a test compound at a concentration and for a period of time that is at least three (3) days; determining the number of adult pluripotent stem cells, exeluding gonadal phosphorylated H3-positive cells, within the first regenerating flatworm after the period of time, wherein the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, is determined with a phosphorylated histone H3 immunoassay; exposing a second regenerating flatworm to a non-carcinogenic, control compound for the period of time that the first regenerating flatworm was exposed to the test compound; and determining the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, within the second regenerating flatworm after treatment with the non-carcinogenic, control compound with a phosphorylated histone H3 immunoassay, wherein the test compound is determined to be a genotoxic carcinogen when the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, determined in the first regenerating flatworm is significantly less than the number of adult pluripotent stem cells, excluding gonadal phosphorylated H3-positive cells, determined in the second regenerating flatworm.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
DETAILED DESCRIPTION
(7) This disclosure will now be illustrated by the following non-limiting examples.
EXAMPLES
Example 1
In Vivo Methods to Detect Whether a Compound is a Non-genotoxic Carcinogen
(8) Material and Methods
(9) Chemicals and Compound Selection Strategy
(10) Cyclophosphamide monohydrate (CP, Cas # 6055-19-2), Cyclosporin A (CA, Cas # 59865-13-3), Diethylstilbestrol (DES, Cas # 56-53-1), D-mannitol (Dmann, Cas # 69-65-8), 4-Nitroquinoline-1-oxide (4NQO, Cas # 56-57-5) and Sulfisoxazole (Sox, Cas # 127-69-5), were all purchased from Sigma Aldrich. These chemicals were selected on the basis of available in vitro and in vivo data reported in the OECD DRP31 document (26), IARC classifications and the publications by Kirkland et al. (13, 27) and Robinson and MacDonald (28). Based on these publications, a distinction was made between genotoxic carcinogens, non-genotoxic carcinogens and non-carcinogens. A carcinogen is defined as any chemical of which the carcinogenic potential has been clearly demonstrated in a two-year rodent bioassay. A genotoxic carcinogen must have shown to be positive in classical in vitro and in vivo genotoxicity assays. CP and 4NQO were selected as non-genotoxic (i.e., requiring metabolization) and genotoxic carcinogens, respectively. A non-genotoxic carcinogen is any chemical of which a non-DNA-reactive mechanism is the primary cause for the observed carcinogenic effects in mammalians and humans. DES and CA were selected as non-genotoxic carcinogens. A compound that is negative in the standard in vitro and in vivo genotoxicity tests and that lacks carcinogenic potential in the two-year mouse and rat bioassays is considered non-carcinogenic. Dmann and Sox were selected as non-carcinogens.
(11) Animal Culture
(12) Cultures of Macrostomum lignano (37) were grown in artificial seawater (ASW) enriched with f/2-medium (40) and fed ad libitum with the diatom Nitzschia curvilineata (PAE culture collection; UGent on the World Wide Web at pae.ugent.be/collection.htm), as described in references 41 and 43.
(13) Test Procedure
(14) One week before starting chemical exposure, a six-well plate filled with 4 ml f/2 medium (Novolab NV) was inoculated with diatoms to achieve an ad libitum food source during exposure. Starvation of the worms leads to a decrease in stem cell proliferation (as shown in Nimeth et al (29)), which would interfere with stem cell proliferation, the endpoint of the assay. Before the start of exposure, fifteen adult worms per condition were pipetted in each well, which was then emptied by pipetting without drying out the worms and refilled with f/2 medium (control), if required, F2+0.05% DMSO (vehicle control) and a concentration of the chemical.
(15) The DMSO concentration in the vehicle control never exceeded 0.05%. This represented the maximum DMSO percentage in the chemical's highest test concentration. Literature data demonstrated that DMSO concentrations greater than 0.1% should be avoided in order to be able to reliably observe any behavioral or toxic effects of compounds in these animals (30-32).
(16) To select the appropriate compound concentration, first, a broad concentration range-finding experiment (spacing factor 10; two weeks exposure) was carried out. Based on this pilot range finding, a fine-tuned range finding (spacing factor less than or equal to 2; for D-Mannitol factor 5) was chosen to conduct the assay. The maximum concentration tested depended on the test compound solubility and the requirement of a survival of twelve out of fifteen worms at the end of exposure.
(17) For all chemicals investigated in this disclosure, the medium was refreshed weekly.
(18) After a preliminary test (CP as reference) on different exposure times ranging from two days to six weeks, the worms were exposed for a period of two weeks, after which they were subjected to phosphorylated histone H3 immunostaining (PH3) to quantify mitosis.
(19) PH3 Immunohistochemistry and Quantification
(20) To quantify mitotic cells in whole mount specimens, the mitotic marker anti-PH3 was used. The staining was performed by first relaxing the animals in a 1:1 mixture of MgCl.sub.2.6H.sub.2O (7.14%) and ASW (5 minutes), then in MgCl.sub.2.6H.sub.2O (7.14%) (undiluted, 5 minutes), followed by fixation in paraformaldehyde (PFA, 4% in PBS, 60 minutes). Animals were then rinsed with phosphate-buffered saline (PBS, 35 minutes) and blocked in bovine serum albumin, containing TRITON (BSA-T, 0.1% TRITON X-100, 60 minutes). Subsequently, animals were incubated in primary rabbit-anti-phos-H3 (1:300 in BSA-T, overnight, 4 C., Upstate Biotechnology), rinsed in PBS (35 minutes) and incubated in secondary TRITC-conjugated swine-anti-rabbit (1:150 in BSA-T, 1 hour, DAKO). Finally, animals were washed with PBS (35 minutes) and slides were mounted using Vectashield (Vector Laboratories). Slides were analyzed using a Leica 2500P microscope equipped with epifluorescence, a 40 objective and a DFC360FX CCD camera.
(21) All somatic PH3-positive cells were counted manually using the Image J cell counter plugin (44) (
(22) Acceptance Criteria and Statistics
(23) The cut-off value of the assay was determined based on density plots (R version 2.15.3; (33)) of the number of mitotic stem cells of all experiments for controls (sea water and DMSO), non-carcinogens, genotoxic carcinogens and non-genotoxic carcinogens. The cut-off value of 30 lays in the tail of the distribution corresponding to the 97.5 percentile or two standard deviations.
(24) Concentrations resulting in the death of 20% of the worms (three out of fifteen) during exposure were considered cytotoxic and were not analyzed. The following criteria were set for a successful test: 1) minimum three independent experiments, 2) the number of mitotic stem cells of two subsequent chemical concentrations must be provided, 3) the number of mitotic stem cells of minimum five worms per concentration must be quantified and finally 4) to account for the possibility of negative control worms exhibiting mitotic numbers exceeding cut-off value, a maximum of two individual control worms (seawater and DMSO) with a number of mitotic stem cells greater than or equal to the cut-off value of 30 is tolerated.
(25) Evaluation Criteria
(26) A compound was considered positive if the following criteria were met: 1) a minimum of 20% of the worms per concentration must have a number of mitotic stem cells greater than or equal to the cut-off value of 30 (scored as +) and 2) at least two out of three of the experiments must have received a + score for the compound to have an overall final + call. In the future, two experiments would be sufficient to determine the overall call, a third experiment would only be required when the number of + and are equal. Plots were generated using the software package R.
(27) Results
(28) During the multistage process of chemical carcinogenesis, intracellular alterations can result in increased cellular proliferation. The endpoint of the in vivo flatworm stem cell proliferation assay is the number of mitotic stem cells. Carcinogens were expected to cause an increase in stem cell proliferation (+ result) and non-carcinogens to have no effect on proliferation after an exposure of two weeks ( result).
(29) A control database was built on the number of mitotic cells per negative control animal. The cut-off value of the assay was determined by the analysis of the density plots of the number of mitotic stem cells for all controls in all experiments (
(30) All controls fulfilled the acceptance criteria of less than two worms per experiment exhibiting a number of mitotic stem cells greater than or equal to the cut-off value. For seawater controls (mean 20.1; n=232) the number of mitotic stem cells was maximally 38 and 50% of the counts were found between 17 and 28 cells. For DMSO controls (mean 21.0; n=89) the number of mitotic stem cells was maximally 35 and 50% of the counts were found between 18 and 24 cells. In all controls, the number of mitotic stem cells were less than 30 with a few exceptions (five out of 232 worms for seawater and four out of 89 in DMSO controls). As such, the cut-off value was set to 30 mitotic stem cells.
(31) Non-carcinogens
(32) D-mannitol was predicted negative by the assay for all three experiments with 0% worms having a number of mitotic stem cells exceeding the cut-off value, even up to 5 mM (910 g/ml) (
(33) Genotoxic Carcinogens
(34) Cyclophosphamide monohydrate was predicted positive in all experiments, each time at 100 M (26 g/ml). One experiment with CP was rejected as only three worms were quantified although 66.7% (100 M) of them had a number of mitotic stem cells exceeding the cut-off value (Table 1).
(35) 4-Nitroquinoline-1-oxide resulted in a positive prediction in the assay for two-thirds of the experiments at both 0.25 (0.047 g/ml) and 0.5 M (0.095 g/ml). At the concentrations predicted positive, the percentage of worms exhibiting a number of mitotic stem cells greater than or equal to the cut-off value ranged from 33.3 to 77.8 (Table 1). In the second experiment that was predicted negative, two subsequent concentrations resulted in a percentage of worms exhibiting a number of mitotic stem cells greater than or equal to the cut-off value, but this percentage was, however, not higher than 20% (Table 1). Higher CP and 4NQO concentrations resulted in greater than 20% lethality of the worms and were not analyzed. Altogether, both compounds were finally predicted positive in the assay (
(36) Non-genotoxic Carcinogens
(37) Diethylstilbestrol was predicted positive for all three experiments at concentrations ranging from 0.3125 M (0.04 g/ml) to 0.625 M (0.1 g/ml). In experiment one, 0.3125 M resulted in 100% of worms exhibiting a number of mitotic stem cells exceeding the cut-off value. In two out of three experiments, even two consecutive concentrations within the range were found positive (
(38) For Cyclosporin A, all experiments fulfilled the acceptance criteria resulting in a positive prediction in the assay. Positive concentrations ranged from 0.125 M (0.14 g/ml) to 0.5 M (0.56 g/ml) with a percentage of mitotic cells greater than or equal to the cut-off value ranging from 36.4% to 70%. Overall, both compounds were predicted positive in the assay (
(39) TABLE-US-00001 TABLE 1 Prediction model. Table representing the number of worms analyzed per concentration (=N), the proportion of worms having 30 or more mitotic stem cells (A), the resulting predictions of all experiments for all tested compounds and the final prediction call. Experiment 1 Experiment 2 Experiment 3 Overall Compound (M) N %.sup.A Pred.sup.B N %.sup.A Pred.sup.B N %.sup.A Pred.sup.B prediction Non-carcinogens 0 6 0 10 0 8 0 D-mannitol .sup.C 1000 8 0 10 0 8 0 5000 8 0 6 0 8 0 Neg Neg Neg Neg Sulfisoxazole .sup.C 0 9 0 6 0 9 0 250 12 0 NA NA NA 9 0 500 9 0 9 11.1 8 0 1000 7 0 10 0 9 0 Neg Neg Neg Neg Genotoxic carcinogen 0 9 0 6 0 6 0 4-Nitroquinoline-1-oxide .sup.C 0.125 NA NA NA 6 16.7 NA NA NA 0.25 6 33.3 + 8 12.5 8 37.5 + 0.5 9 77.8 + 7 0 11 36.4 + Pos Neg Pos Pos Cyclophosphamide 0 8 0 9 0 11 0 monohydrate .sup.C 50 6 0 8 0 12 16.7 100 .sup.3 .sup.D 66.7 NA 10 20 + 6 33.3 + NA Pos Pos Pos Non-genotoxic carcinogen 0 10 0 NA NA NA 8 0 Diethylbestrol .sup.E DMSO 8 0 6 0 8 0 0.3125 10 100 + 8 12.5 12 33.3 + 0.625 11 27.3 + 10 50 + 9 22.2 + Pos Pos Pos Pos Cyclosporine A .sup.E 0 12 0 8 0 9 0 DMSO 12 0 6 0 6 0 0.125 NA NA NA 7 0 8 37.5 + 0.25 10 70 + 7 0 7 42.9 + 0.5 10 40 + 11 36.4 + 8 50 + Pos Pos Pos Pos When 20% of the number of mitotic cells in an experiment exceeded the cut-off value of 30, a + was scored for that experiment. One + per experiment was required for predicting the compound as positive (=Pos) and a minimum of two out of three experiments with positive predictions resulted in a final positive prediction call for that compound (see evaluation criteria in Material and Methods). Concentrations that were not analyzed because of experimental loss during test procedure or rejected because of test invalidity (for example, N < 3 for CP) were coded as not applicable (NA). Dmann: D-Mannitol; Sox: Sulfisoxazole; Nqo: 4-nitroquinoline-1-oxide; Cp: cyclophosphamide monohydrate; Des: diethylstillbestrol; CA: cyclosporine A. .sup.Aproportion of worms having 30 or more mitotic stem cells .sup.B+ in case 20% or more worms having 30 or more mitotic stem cells .sup.C in Seawater (SW) .sup.D Less than five worms were quantified for this concentration, this experiment was excluded. .sup.E DMSO conc 0.05%
Test Organism
(40) Asexual strains of the freshwater planarian Schmidtea mediterranea (38, 45) were maintained in culture medium, consisting of water, that was first deionized and then distilled, and to which the following nutrients were added: 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl and 1.2 mM NaHCO3. The animals were continuously kept in the dark at a room temperature of 20 C. and were fed once a week with veal liver.
(41) Experimental Design
(42) Experiments were performed with regenerating animals (heads and tails) to accelerate initial responses to carcinogenic compounds, as this process of massive cell proliferation, migration and differentiation resembles the process of carcinogenesis, and, possibly differently affects the dynamics of stem cells toward genotoxic and non-genotoxic carcinogen exposures (16, 32, 39).
(43) In each experimental condition, animals were cut transversally immediately before exposure to a genotoxic, non-genotoxic or non-carcinogen concentration, medium (control) or medium with DMSO (vehicle control). Exposures were performed in six-well plates (4 ml/well). To exclude intrinsic effects of DMSO on cell proliferation, the DMSO vehicle concentration never exceeded 0.05% and interaction effects between the test compound and DMSO were investigated using a two-way ANOVA (32). Compound selection was based on literature screens (13, 26-28) and included the genotoxic carcinogens methyl methane sulphonate (MMS; Cas # 66-27-3; purity 99%) and 4-nitroquinoline-1-oxide (4NQO; Cas # 56-57-5; purity 98%), the non-genotoxic carcinogens methapyrilene hydrochloride (MPH; 135-23-9; analytical standard), cyclosporine A (CsA; Cas # 59865-13-3; purity 98.5%), chlorpromazine hydrochloride (CPZ; Cas # 69-09-0; purity 98%) and sodium phenobarbital (S-PB; Cas # 57-30-7) and the non-carcinogen D-mannitol (Dmann, Cas # 69-65-8), all purchased from Sigma-Aldrich (Saint Louis, Mo., USA). After comparing continuous versus intermittent (repeat-dose toxicity: two days of compound exposure, three days of recovery in culture medium) exposures and medium refreshment once versus twice a week, a continuous exposure with medium refreshment twice a week was found most suitable to obtain distinctive cellular responses. Animals were fed once a week with veal liver and starved for one week before stem cell proliferation measurements. Stem cell proliferation was measured after one day of regeneration (the mitotic minimum of regeneration), after three days (the mitotic maximum of regeneration), after one week (the end stage of regeneration) after two and six weeks (during homeostasis), as well as at the appearance of organismal phenotypes (15, 46, 47). Phenotypic effects were monitored daily with a stereo microscope.
(44) Mitotic Activity of Stem Cells
(45) Stem cells are the only proliferating cells in these animals (48). Observed changes in cell proliferation can thus be directly linked to stem cell responses. The mitotic activity of stem cells was determined by immunostaining with anti-P-Histone H3 antibody (Millipore), performed as previously described (32, 49). After exposure, the worms were treated for 5 minutes on ice with five-eighths Holtfreter (50) solution containing 2% HCI (VWR) to remove the mucus layer. The samples were fixed in Carnoy's fixative (51) for 2 hours (on ice) and were rinsed in 100% methanol (VWR) during 1 hour and bleached overnight at room temperature in 6% H.sub.2O.sub.2 (VWR) (in 100% methanol). Subsequently, the worms were rehydrated through a graded series of methanol/phosphate buffered saline-triton (PBST) (PBS tablets; VWR) (TRITON X-100 pro analyse, VWR) washes (75%, 50%, 25%, 0%) for 10 minutes each, where after non-specific binding sites were blocked in bovine serum albumin (Sigma Aldrich) (PBST/BSA) (0.1% TRITON X-100, and 0.1 g/ml BSA) for 3 hours. Animals were incubated at 4 C. for 44 hours with a primary antibody (anti-phospho-Histone H3 (Ser10), biotin conjugate, Millipore, catalogue number: 16-189) 1:600 diluted in PBST/BSA. The animals were rinsed repeatedly for 1 hour in PBST and incubated in PBST/BSA for 7 hours. Then, the animals were incubated with a secondary antibody (goat anti-rabbit IgG rhodamine conjugated, Millipore, catalogue number: 12-510), 1:500 diluted in PBST/BSA for 16 hours. Afterwards, animals were rinsed repeatedly for 30 minutes in PBST and mounted in glycerol. The animals were examined with fluorescence microscopy performed with a Nikon Eclipse 80i. The total number of mitotic neoblasts was normalized to the body size of the animals (cfr. Body area). Before the start of the coloring, three photos were taken of each animal at the moment the body was fully stretched. For normalization, of these three photos, the average body size was calculated, using Image J (1.44p, National Institutes of Health).
(46) Phenotypic Follow-up
(47) An in vivo study allows the investigation of the entire organism's response to carcinogenic compounds and, as such, makes it possible to link carcinogen-induced stem cell proliferation with the organism's phenotype. Conditions were subdivided according to their phenotypic effects into concentrations that caused no observable phenotypic effects (category 1), phenotypic effects (category 2, described in phenotypic effect screening) and concentrations that induced organismal death (category 3). Phenotypic parameters that were monitored daily were tissue regression, lesion, blisters, bloating, pigmentation, behavior, regenerative success, body size, tissue outgrowth, flattened posture and organismal death (35). Stem cell proliferation was quantified at the appearance of systemic effects (category 2 concentrations).
(48) Statistical Methods
(49) Effects of time and carcinogenic exposures (including low and high concentrations of carcinogens) on the number of dividing cells were analyzed using an ANOVA model including time, exposure and the interaction between both covariates (
(50) Results
(51) Stem cell proliferation was monitored at physiological meaningful time points after exposure to both low and high concentrations of genotoxic, non-genotoxic and non-carcinogens (
(52) To assess if the observed differences in cell proliferation after one and three days of exposure are not compound-specific responses, and as such can be generalized to genotoxic and non-genotoxic carcinogens, the organisms were exposed to another genotoxic (4NQO) and non-genotoxic (S-PB) carcinogen (
(53) Phenotypic Effects and Associated Stem Cell Responses.
(54) To further distinguish between carcinogen and non-carcinogen exposures and between genotoxic and non-genotoxic carcinogens, phenotypic effects as a discriminative factor were studied. Exposure concentrations were subdivided into categories, in which category 1 consisted of concentrations that induced no phenotypic effects, category 2 consisted of concentrations that induced phenotypic effects and category 3 were lethal concentrations (Table 2).
(55) TABLE-US-00002 TABLE 2 Classification of genotoxic and non-genotoxic carcinogen concentrations according to observed phenotypes. Toxicity Carcinogens Cat. 1 Cat. 2 Cat. 3 Genotoxic MMS 25-50-100-200 M 400-500 M 1 mM 4NQO 0.1-0.5 M 2 M 5 M Non-genotoxic S-PB 1 mM 2 mM 4 mM CPZ 250-500 nM 750-1000 nM 2 M MPH 10 M 150-250 M 500 M Concentrations of genotoxic (MMS, 4NQO) and non-genotoxic (S-PB, CPZ, MPH) carcinogens were classified into three categories according to the phenotypic effects they induced: Category 1: No effects on phenotype; Category 2: Phenotypic effects; Category 3: Mortality. For category 2 concentrations in bold, stem cell proliferation responses are presented in the article. Exposure times were three days (MMS), one week (4NQO) and two weeks (S-PB, CPZ, MPH). MMS: methyl methane sulphonate; 4NQO: 4-nitroquinoline-1-oxide; S-PB: sodium phenobarbital; CPZ: chlorpromazine hydrochloride; MPH: methapyrilene hydrochloride
(56) In contrast to carcinogen exposures, no phenotypic effects were induced by non-carcinogen (Dmann) exposures. Category 2 concentrations of genotoxic carcinogens induced phenotypic effects such as aberrant pigmentation spots, severe tissue regression, regeneration failures and aberrant behavior (Table 3).
(57) TABLE-US-00003 TABLE 3 Number of animals with specific phenotypic effects after exposure to category 2 concentrations of genotoxic and non-genotoxic carcinogens (see Table 2). GTX NGTX Phenotypes MMS 4NQO S-PB CPZ MPH Tissue regression 3/14 2/4 / / / Pigmentation 1/14 2/4 / / / Behavior 14/14 3/4 2/2 4/4 8/8 Regenerative success 14/14 4/4 / / / Body size 10/14 / / 2/2 8/8
(58) An exposure to category 2 concentrations of non-genotoxic carcinogens elicited physiological responses, which initially seemed to have a smaller impact on the organism's viability in comparison with genotoxic carcinogen-induced phenotypes, e.g., changes in body size or length or an aberrant behavior. Interestingly, a quantification of cell proliferation at the appearance of phenotypic effects further discriminated genotoxic and non-genotoxic carcinogen exposures: Genotoxic carcinogen-induced phenotypic effects (category 2 concentrations) were associated with a significant drop in stem cell proliferation compared to non-exposed animals, while no proliferation changes were observed at the manifestation of non-genotoxic carcinogen-induced phenotypes (
REFERENCES
(59) 1. EU (2008) 440/2008 of 31 May 2008 laying down test methods pursuant to Regulation (EC) 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH), ed (EC) CR (Official Journal of the European Union, L 142/1-L 142/739). 2. Marone P. A., Hall W. C., and Hayes A. W. (2014). Reassessing the two-year rodent carcinogenicity bioassay: a review of the applicability to human risk and current perspectives. Regulatory toxicology and pharmacology: RTP 68(1):108-118. 3. Heinonen T., Louekari K., and Thti H. (2014). Need for Harmonized Strategies and Improved Assessment of Carcinogenic and Genotoxic Potencies of Chemical Substances. Journal of Translational Toxicology 1(1):76-87. 4. ICCVAM (2000) ICCVAM Authorization Act of 2000 Public Law 106-545. 5. Doktorova T. Y., Pauwels M., Vinken M., Vanhaecke T., and Rogiers V. (2012). Opportunities for an alternative integrating testing strategy for carcinogen hazard assessment? Critical Reviews in Toxicology 42(2):91-106. 6. Ellinger-Ziegelbauer H., Stuart B., Wahle B., Bomann W., and Ahr H. J. (2005). Comparison of the expression profiles induced by genotoxic and nongenotoxic carcinogens in rat liver. Mutat. Res. 575(1-2):61-84. 7. Hernndez L. G., van Steeg H., Luijten M., and van Benthem J. (2009). Mechanisms of non-genotoxic carcinogens and importance of a weight of evidence approach. Mutation Research/Reviews in Mutation Research 682(2-3):94-109. 8. Silva Lima B. and Van der Laan J. W. (2000). Mechanisms of Nongenotoxic Carcinogenesis and Assessment of the Human Hazard. Regulatory Toxicology and Pharmacology 32(2):135-143. 9. Bogdanffy M. S. and Valentine R. (2003). Differentiating between local cytotoxicity, mitogenesis, and genotoxicity in carcinogen risk assessments: the case of vinyl acetate. Toxicology Letters 140-141(0):83-98. 10. van Delft J. H., et al. (2004). Discrimination of genotoxic from non-genotoxic carcinogens by gene expression profiling. Carcinogenesis 25(7):1265-1276. 11. Adler S., et al. (2011). Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch. Toxicol. 85 (5):367-485. 12. Benigni R., Bossa C., and Tcheremenskaia O. (2013). In vitro cell transformation assays for an integrated, alternative assessment of carcinogenicity: a data-based analysis. Mutagenesis 28(1):107-11 6. 13. Kirkland D., Aardema M., Henderson L., and Mller L. (2005). Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 584(1-2):1-256. 14. Robb S. M. C., Ross E., and Alvarado A. S. (2008). SmedGD: the Schmidtea mediterranea genome database. Nucleic Acids Research 36 (suppl 1):D599-D606. 15. Dunkel J., Talbot J., and Schotz E. M. (2011). Memory and obesity affect the population dynamics of asexual freshwater planarians. Phys. Biol. 8(2):026003. 16. Alexandrov V., Aiello C., and Rossi L. (1990). Modifying factors in prenatal carcinogenesis (review). In Vivo 4(5):327-335. 17. Birnbaum L. S. and Fenton S. E. (2003). Cancer and developmental exposure to endocrine disruptors. Environmental health perspectives 111(4):389-394. 18. Oviedo N. J. and Beane W. S. (2009). Regeneration: The origin of cancer or a possible cure? Seminars in Cell and Developmental Biology 20(5):557-564. 19. Hanahan D. and Weinberg Robert A. (2011). Hallmarks of Cancer: The Next Generation. Cell 144(5):646-674. 20. Best J. B. and Morita M. (1982). Planarians as a model system for in vitro teratogenesis studies. Teratogenesis, carcinogenesis, and mutagenesis 2(3 -4):277-291. 21. Hall F., Morita M., and Best J. B. (1986). Neoplastic transformation in the planarian: I. Cocarcinogenesis and histopathology. The Journal of experimental zoology 240(2):211-227. 22. Schaeffer D. J. (1993). Planarians as a model system for in vivo tumorigenesis studies. Ecotoxicology and environmental safety 25(1):1-18. 23. Hall F., Morita M., and Best J. B. (1986). Neoplastic transformation in the planarian: II. Ultrastructure of malignant reticuloma. The Journal of experimental zoology 240(2):229-244. 24. Fu Z. and Tindall D. J. (2008). FOXOs, cancer and regulation of apoptosis. Oncogene 27(16):2312-2319. 25. Stalmans S., et al. (2014). Flatworm models in pharmacological research: the importance of compound stability testing. Regulatory toxicology and pharmacology: RTP 70(1):149-154. 26. OECD OfECaD (2007). Detailed review on cell transformation assays for detection of chemical carcinogens. OECD Environment, Health and Safety Publications, Series on Testing and Assessment No. 31. 27. Kirkland D., Kasper P., Muller L., Corvi R., and Speit G. (2008). Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: A follow-up to an ECVAM workshop. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 653(1-2):99-108. 28. Robinson D. E. and Macdonald J. S. (2001). Background and framework for ILSI's collaborative evaluation program on alternative models for carcinogenicity assessment. Toxicologic pathology 29 (1 suppl):13-19. 29. Nimeth K. T., et al. (2004). Stem cell dynamics during growth, feeding, and starvation in the basal flatworm Macrostomum sp. (Platyhelminthes). Developmental dynamics 230(1):91-99. 30. Pagan O. R., Rowlands A. L., and Urban K. R. (2006). Toxicity and behavioral effects of dimethylsulfoxide in planaria. Neuroscience letters 407(3):274-278. 31. Yuan Z., Zhao B., and Zhang Y. (2012). Effects of dimethylsulfoxide on behavior and antioxidant enzymes response of planarian Dugesia japonica. Toxicology and industrial health 28(5):449-457. 32. Stevens A. S., et al. (2014). Toxicity profiles and solvent-toxicant interference in the planarian Schmidtea mediterranea after dimethylsulfoxide (DMSO) exposure. Journal of applied toxicology: JAT. 33. Team R D C (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 34. Newmark P. A., Reddien P. W., Cebria F., and Alvarado A. S. (2003). Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proceedings of the National Academy of Sciences 100 (suppl 1):11861-11865. 35. Reddien P. W., Bermange A. L., Murfitt K. J., Jennings J. R., and Sanchez Alvarado A. (2005). Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Developmental cell 8(5):635-649. 36. Ladurner P., Rieger R., and Baguna J. (2000). Spatial distribution and differentiation potential of stem cells in hatchlings and adults in the marine platyhelminth macrostomumsp.: a bromodeoxyuridine analysis. Dev. Biol. 226(2):231-241. 37. Ladurner P., Scharer L., Salvenmoser W., and Rieger R. M. (2005). A new model organism among the lower Bilateria and the use of digital microscopy in taxonomy of meiobenthic Platyhelminthes: Macrostomum lignano, n. sp. (Rhabditophora, Macrostomorpha) Ein neuer Modellorganismus fr die basalen Bilaterier und die Verwendung von digitaler Mikroskopie fr die Taxonomie meiobenthischer Platyhhelminthen: Macrostomum lignano n. sp. (Rhabditophora, Macrostomorpha). Journal of Zoological Systematics and Evolutionary Research 43(2):114-126. 38. Benazzi M., Baguna J., Ballester R., Puccinelli I., and Papa R. D. (1975). Further Contribution to the Taxonomy of the Dugesia Lugubris-Polychroa Group
with Description of Dugesia Mediterranea N. SP. (Tricladida, Paludicola). Bolletino di zoologia 42(1):81-89. 39. Newmark P. A. and Sanchez Alvarado A. (2000). Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Dev. Biol. 220(2):142-153. 40. Guillard R. R. L. and Ryther J. H. (1962). STUDIES OF MARINE PLANKTONIC DIATOMS: I. CYCLOTELLA NANA HUSTEDT, AND DETONULA CONFERVACEA (CLEVE) GRAN. Canadian Journal of Microbiology 8(2):229-239. 41. Rieger R., et al. (1988). Laboratory cultures of marine Macrostomida (Turbellaria). Fortschr. Zool. 36:523. 42. Ladurner P., et al. (2008). The stem cell system of the basal flatworm Macrostomum lignano. Stem cells (Springer), pp 75-94. 43. Mouton S., et al. (2009). The free-living flatworm Macrostomum lignano: a new model organism for ageing research. Experimental gerontology 44(4):243-249. 44. Abramoff M. D., Magalhaes P. J., and Ram S. J. (2004). Image processing with ImageJ. Biophotonics international 11(7):36-43. 45. Baguna J. (1973). Estudios citotaxonomicos, ecologicos e histofisiologfa de la regulation morfogenetica durante el crecimiento y la regeneration en la raza asexuada de la planaria Dugesia mediterranea n. sp. (Turbellaria: Tricladida: Paludicola). 46. Robb S. M., Ross E., and Sanchez Alvarado A. (2008). SmedGD: the Schmidtea mediterranea genome database. Nucleic acids research 36(Database issue):D599-606. 47. Salo E. and Baguna J. (1984). Regeneration and pattern formation in planarians. I. The pattern of mitosis in anterior and posterior regeneration in Dugesia (G) tigrina, and a new proposal for blastema formation. Journal of embryology and experimental morphology 83:63-80. 48. Reddien P. W. and Alvarado A. S. (2004). Fundamentals of planarian regeneration. Annual review of cell and developmental biology 20:725-757. 49. Plusquin M., et al. (2012). Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea. The International journal of developmental biology 56(1-3):183-191. 50. Armstrong J. B., Duhon S. T., and Malacinski G. M. (1989). Raising the axolotl in captivity. Smith VII. 51. Yoneyama M., Kitayama T., Taniura H., and Yoneda Y. (2003). Immersion fixation with Carnoy solution for conventional immunohistochemical detection of particular N-methyl-D-aspartate receptor subunits in murine hippocampus. Journal of neuroscience research 73(3):41 6-426. 52. Doktorova T Y, et al. (2014). Testing chemical carcinogenicity by using a transcriptomics HepaRG-based model? 53. Hochstenbach K, et al. (2012). Transcriptomic fingerprints in human peripheral blood mononuclear cells indicative of genotoxic and non-genotoxic carcinogenic exposure. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 746(2):124-134.