
Many
We have been making attempts to introduce
The rate of concordance between the MOT assay results and the
Chemicals other than in-house candidates were of reagent grade.
Ethanol, dimethyl sulfoxide, chlorpromazine, aspirin, cyclophosphamide, 6-amino nicotinamide, diethyl stilbestrol, phenytoin, cortisone, aminopterin, all-trans retinoic acid, and 5-fluorouracil were used as developmentally toxic compounds. Saccharin, amaranth, L-ascorbic acid, L-glutamic acid, diphenhydramine, histamine, isoniazid, and pyridoxine were used as non-developmentally toxic compounds.
Strong embryotoxic compounds included methotrexate, all-trans retinoic acid, 6 aminonicotinic acid, bromodeoxyuracil, and hydroxyurea. Weak embryotoxic compounds included lithium, boric acid, valproic acid, dimethadione, salicylic acid, and methoxyacetic acid. Non-embryotoxic compounds included penicillin G, saccharin, acrylamide, camphor, diphenhydramine, and dimethylphthalate.
Test chemicals were supplied by Sumitomo Chemical Co., Ltd.
MOT assay was conducted using a previously described method (11). Procedures involving animals and their care were in compliance with Japanese law and were approved by our company’s IACUC.
Disks (1.25 cm diameter) were cut from polyethylene sheets. The disks were floated overnight at room temperature on a solution of PBS containing 2.5% glutaraldehyde and 50 μg/mL concanavalin A, washed in PBS, and stored in 10 mM Tris-HCl (pH 7.4) containing 0.3 M glycine, 1 mM manganese chloride, and 1 mM sodium azide. Bovine serum albumin coated disks served as non-attachment controls.
Female ICR mice purchased from Japan SLC, Inc. (Shizuoka, Japan) were inoculated with 5 × 105 ascitic mouse tumor cells (Ehrlich ascites cells from Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan) about 2 weeks before use. The cells were harvested, washed in PBS, and re-suspended in Eagle’s minimal essential medium with 5% fetal bovine serum. For radioactive labeling, the cells were incubated with 12.5 μCi 3H thymidine per 108 cells at 37°C for 1 hr with gentle agitation. After washing three times in PBS, aliquots (1 mL) of labeled cells at 107 cells/mL were incubated with various concentrations of the test agent for 30 min at 37°C. Test solutions were prepared by serial dilution. The maximum concentration was set at a concentration that is lethal to a majority of the cells or the maximum water soluble concentration.
Three or four disks were placed in a plastic Petri dish (35 mm diameter) with lectin-coated side up, permitting adhesion of the disks to the bottom surface of the dish. The cells were poured into the dish and allowed to sediment onto the disks for 20 min. The disks were then removed with fine forceps, washed in PBS, and radioassayed. The radioactivity of the disks was quantified by conventional liquid scintillation counting with a Packard Tri-Carb 460 CD. The cell suspensions were incubated with 0.1% trypan blue and examined for the presence of dead cells. Dead cells do not attach to lectin-coated surfaces, hence, detached trypan-blue positive cells were not considered to be the product of “true” inhibition.
The adherent cell count on the bovine serum albumin-coated disks (non-attachment control) was subtracted from the average adherent cell count on concanavalin A-coated disks. The results were plotted as a percentage of attachment of vehicle-treated cells to concanavalin A-coated disks. More than two experimental runs were conducted for each chemical. When the percentages of the attached cells exposed to a chemical were lower than those of cell viability, the chemical was judged to inhibit the attachment of viable cells and classified as positive.
EST assay was conducted as previously described (18) with modification. Each test chemical was classified into three categories based on published criteria (18).
BALB/c 3T3 (American Type Culture Collection, VA, USA) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 4 mM glutamine, 100 U penicillin/mL, and 0.1 mg streptomycin/mL. Embryonic stem (ES) cells (American Type Culture Collection) were maintained in serum-free media for mouse ES cells with 0.1% mercaptoethanol, and 1,000 U leukemia inhibitory factor (LIF)/mL. As differentiation medium, DMEM containing l% non-essential amino acids, 0.1% mercaptoethanol, 100 U penicillin/mL, 0.1 mg streptomycin/mL, and 15% fetal bovine serum was used.
A thousand cells were seeded into each well of a 96-well plate and grown in the presence of a concentration range of a test chemical diluted in culture medium in the absence of LIF. After 4 days of culture, the viability of the cells was determined using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). The cells were treated with different concentrations of the test chemicals and the cytotoxicity for each cell line was expressed as the concentration inhibiting growth by 50% of the control level (IC503T3 and IC50ES).
Four hundred undifferentiated ES cells in droplets of 20 μL were seeded onto the bottom of the lid of a culture dish and grown for 3 days in hanging drop culture in the presence of a concentration range of test chemical diluted in differentiation medium to allow differentiation into embryoid bodies (EBs). After 3 days in hanging drop culture, the EBs were transferred to Petri dishes containing differentiation medium with the test chemical for another 2 days. On day 5, the EBs were seeded singly into one well of a 24-well tissue culture plate (containing the test chemical) to allow adherence and outgrowth of the EBs, and the development of contracting cardiomyocytes. Differentiation was assessed by microscopic inspection of the EBs on day 10. The percentage of EBs developing contracting cardiomyocytes was determined and compared to that in the control. The EBs were treated with different concentrations of the test chemicals and the inhibition of differentiation is expressed as the concentration of test chemical that inhibited the development of contracting cardiomyocytes by 50% of the control (ID50).
Previously described prediction model consisting of linear discriminant functions and classification criteria (18) was used. Endpoints (ID50, IC50ES, and IC503T3) of test chemicals were applied as variables in the linear discriminant functions and, then, embryotoxicity of each test chemical was classified as non, weak or strong according to the classification criteria. Linear discriminant functions are expressed in the following equations:
When the result of function I exceeds those of functions II and III, the chemical is classified as non-embryotoxic. When the function II result is larger than those of functions I and III, the chemicals is weakly embryotoxic. When the function III result is the largest, the chemical is strongly embryotoxic.
The principle of the MOT assay is to identify chemicals that interfere with energy-dependent attachment of tumor cells to the concanavalin A-coated surfaces of plastic disks. Cell-to-cell or cell-to-extracellular matrix interaction plays a key role in development. Cell-cell interaction during embryogenesis requires metabolic energy. Tumor cell attachment to concanavalin A may mimic cell-cell interactions in embryos. This possible model of cell-cell interactions in embryos was used in the
As the first step towards introducing the assay into our facility, we studied the MOT assay using existing chemi346 cals to determine its suitability as a screen for developmental toxicity. Data regarding the
With 99 chemicals including existing chemicals, in-house chemicals, and a group of structurally, bioactively similar candidate chemicals, the overall concordance rate between the MOT assay in our facility and
The EST assay was initially conducted with 17 chemicals that were used in the ECVAM validation study (20). The concordance rate was 65% (Table 2). The strong embryotoxic chemicals were well detected. However, half of the weak embryotoxic chemicals
We analyzed the results of the EST assay to determine why the EST assay tends to over-classify chemicals as weakly toxic. We noticed that EST classification might be, in part, explained by cell growth inhibition. The results of sorting ECVAM validation of the test chemicals by IC50ES revealed that classification by the prediction model (18) might be consistent with the degree of cell growth inhibition (Table 3). This may reflect the relationship between the
We examined the characteristics of the prediction model by using simple fictitious IC50 and ID50 values for classification purpose (Table 5). To investigate a possible relationship between IC50ES (cell growth) and ID50 (cell differentiation), we classified chemicals according to the prediction model and used various ID50 values with IC50ES and IC503T3 values fixed at the median of each concentration range for sorting (left column of Table 5). Chemicals with IC50ES in the range of 0.1 to 1, 1 to 10, and 10 to 100 μg/mL were classified as non-toxic by the prediction model when the ID50/IC50ES ratio was higher than 5, 3, and 2, respectively. Thus, it was inferred that chemicals with IC50ES in the range of 0.1 to 100 μg/mL should have an ID50/IC50ES ratio of 2 or more in order not to be classified as weak or strong by the prediction model. Similarly, a possible relationship between mature 3T3 and immature ES cells was investigated with fixed IC50ES and ID50 values, and various IC503T3 values (right column of Table 5). It was also suggested that chemicals with IC50ES in the range of 0.1 to 100 μg/mL might be classified as non-toxic when the IC503T3/IC50ES ratio was lower than 0.5. Accordingly, it is likely that the prediction model classifies a chemical as weak or strong when the ID50/IC50ES ratio is lower than 2 and the IC503T3/IC50ES ratio is higher than 0.5. The IC50 and ID50 values of test chemicals may need to differ by a considerable amount for chemicals to be classified as non-toxic. We found that IC50ES values of the in-house chemicals were close to their ID50 and IC503T3 values (Table 6). The ratios of ID50/IC50ES or IC503T3/IC50ES ranged from 0.5 to 2 in a majority of the in-house chemicals tested. Therefore, the in-house chemicals might be classified as weakly toxic rather non-toxic. When the data of the ECVAM validation test were analyzed, the values of the parameters were also close to one another.
We conducted cell-based developmental toxicity assays devised two decades apart and investigated their characteristics. The results indicated that both assays had high false positive rates. Chemicals tested included agrochemicals such as fungicides, insecticides, and herbicides; pharmaceuticals targeting the nervous, cardiovascular, and immune systems; and general chemical substances. In the MOT assay, there was little tendency that classification was related to chemical properties. The fact that a group of structurally and bioactively similar chemicals were classified as positive or negative almost at random might also suggest no particular relationship between chemical property and classification
Basically, an
In addition, the selection of reference chemicals as developmental toxicants may be another important issue affecting the inaccuracy of prediction and false positives. The principles of MOT and EST assays probably apply to only a very small number of pathogenetic mechanisms of developmental toxicity (Fig. 2) even when the assay principle is based on real mechanisms of developmental toxicity in animals. We pursued, however, to detect all developmental toxicants with the MOT or EST assay. We evaluated the performance of the MOT and EST assays with a variety of chemicals which induce developmental toxicity by different pathogenetic mechanisms.
Not a few
The concordance rates for the MOT and EST assays were at the 60% level. In dichotomizing test agents into positive and negative, the base rate of concordance (in the case of classification completely by chance) is 50%, not 0%, because a random answer to a binary-choice (yes or no) question is coincidentally correct 50% of the time. A test with concordance rate of 60–70% or perhaps 80% may not have special significance as a screen for candidate chemicals, and for ranking agents for follow-on
Whether a high false positive rate in an
The authors would like to thank Dr. Kunifumi Inawaka and the staff of Sumitomo’s Developmental and Reproductive Biology Team for their excellent assistance.
Comparison of
Test chemicals | Concordance | ||||
---|---|---|---|---|---|
Inhibitory | Noninhibitory | Total | |||
Existing chemicals | Developmentally toxic | 10 | 2 | 12 | 65% |
Developmentally nontoxic | 5 | 3 | 8 | ||
Total | 15 | 5 | 20 | ||
In-house chemicals | Developmentally toxic | 15 | 4 | 19 | 64% |
Developmentally nontoxic | 11 | 12 | 23 | ||
Total | 26 | 16 | 42 | ||
A group of structurally, bioactively similar chemicals | Developmentally toxic | 13 | 10 | 23 | 51% |
Developmentally nontoxic | 8 | 6 | 14 | ||
Total | 21 | 16 | 37 | ||
All chemicals tested | Developmentally toxic | 38 | 16 | 54 | 60% |
Developmentally nontoxic | 24 | 21 | 45 | ||
Total | 62 | 37 | 99 |
Comparison of
Test chemicals | Concordance | ||||
---|---|---|---|---|---|
Strong | Weak | Non | |||
ECVAM validation chemicals | Strong | 5 | 0 | 0 | 65% |
Weak | 0 | 3 | 3 | ||
Non | 0 | 3 | 3 | ||
In-house chemicalsa | Developmentally toxic | 0 | 11 | 0 | |
Developmentally nontoxic | 2 | 19 | 0 |
Many of in-house chemicals were investigated for developmental toxicity in one species (rats), and could not be classified according to Genshow
Distribution of
IC50ES (μg/mL) | Number of test chemicals | ||||||
---|---|---|---|---|---|---|---|
Strong | Weak | Non | Strong | Weak | Non | ||
< 0.1 | 3 | 2 | 0 | 1 | 3 | 0 | 0 |
0.1–1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 |
1–10 | 2 | 2 | 0 | 0 | 2 | 0 | 0 |
10–100 | 3 | 0 | 3 | 0 | 0 | 1 | 2 |
100–1000 | 9 | 0 | 6 | 3 | 0 | 6 | 3 |
> 1000 | 2 | 0 | 0 | 2 | 0 | 0 | 2 |
Data were retrieved from Appendix 2 (laboratory code I) of the publication by Genschow
Distribution of
IC50ES (μg/mL) | Number of test chemicals | |||||
---|---|---|---|---|---|---|
Strong | Weak | Non | Developmentally toxic | Developmentally nontoxic | ||
< 0.1 | 0 | 0 | 0 | 0 | 0 | 0 |
0.1–1 | 2 | 2 | 0 | 0 | 0 | 2 |
1–10 | 6 | 0 | 6 | 0 | 1 | 5 |
10–100 | 22 | 0 | 22 | 0 | 9 | 13 |
100–1000 | 2 | 0 | 2 | 0 | 1 | 1 |
> 1000 | 0 | 0 | 0 | 0 | 0 | 0 |
Analysis of the characteristics of the prediction model of EST assay with fictitious values of IC50s
IC50ES (μg/mL) | Fictitious IC50s | ID50/IC50ES | Fictitious IC50s | IC503T3/IC50ES | ||||||
---|---|---|---|---|---|---|---|---|---|---|
IC503T3 fixed | IC50ES fixed | ID50 varied | IC503T3 varied | IC50ES fixed | ID50 fixed | |||||
0.1–1 | 0.5 | 0.5 | 0.5 | 1 | Strong | 0.1 | 0.5 | 0.5 | 0.2 | Non |
0.5 | 0.5 | 1 | 2 | Strong | 0.125 | 0.5 | 0.5 | 0.25 | Weak | |
0.5 | 0.5 | 2 | 4 | Weak | 0.25 | 0.5 | 0.5 | 0.5 | Strong | |
0.5 | 0.5 | 2.5 | 5 | Non | 0.5 | 0.5 | 0.5 | 1 | Strong | |
1–10 | 5 | 5 | 5 | 1 | Weak | 1.25 | 5 | 5 | 0.25 | Non |
5 | 5 | 10 | 2 | Weak | 2.5 | 5 | 5 | 0.5 | Weak | |
5 | 5 | 15 | 3 | Non | 5 | 5 | 5 | 1 | Weak | |
10–100 | 50 | 50 | 50 | 1 | Weak | 12.5 | 50 | 50 | 0.25 | Non |
50 | 50 | 100 | 2 | Non | 25 | 50 | 50 | 0.5 | Weak | |
50 | 50 | 50 | 1 | Weak | ||||||
100–1000 | 500 | 500 | 450 | 0.9 | Weak | 500 | 500 | 500 | 1 | Non |
500 | 500 | 500 | 1 | Non | 560 | 500 | 500 | 1.1 | Weak |
Comparison of the ID50 and IC503T3 with the IC50ES of in-house chemicals at Sumitomo Chemical Co. LTD by IC50ES concentration range
IC50ES (μg/mL) | Number of test chemicals | Distribution of ratios of in-house chemicals tested at Sumitomo Chemical | |||||||
---|---|---|---|---|---|---|---|---|---|
ID50/IC50ES ratio | IC503T3/IC50ES ratio | ||||||||
0.1–0.5 | 0.5–1 | 1–2 | 2–4 | 0.1–0.5 | 0.5–1 | 1–2 | 2–4 | ||
< 0.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
0.1–1 | 2 | 0 | 0 | 2 | 0 | 0 | 1 | 0 | 1 |
1–10 | 6 | 1 | 1 | 3 | 1 | 0 | 0 | 5 | 1 |
10–100 | 22 | 5 | 13 | 4 | 0 | 1 | 9 | 12 | 0 |
100–1000 | 2 | 0 | 1 | 1 | 0 | 0 | 0 | 2 | 0 |
> 1000 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |