The developmental toxicities of five test compounds including carbon tetrachloride, urethane, phenacetin, parathion. and chloroform, were evaluated using Frog Embryo Teratogenesis Assay - Xenopus (FETAX), with minor modification. Post-isolation mixtures of differently induced rat liver microsomes (phenobarbital- (PB), beta -naphthoflavone(beta -NF), and isoniazid-(INH)-induced preparations) were co-cultured directly with X. laevis embryos. Results from these studies suggest that the Aroclor 1254-induced MAS could effectively be replaced by a mixed lot of PB-, beta -NF-, and INH-induced rat liver microsomes. Each of the test materials were found to be developmentally toxic when bioactivated by the mixed MAS.

FETAX (Frog Embryo Teratogenesis Assay-Xenopus) is a 96-h whole-embryo developmental toxicity screening assay that can be used in ecotoxicology and in detecting mammalian developmental toxicants when an in vitro metabolic activation system is employed. A standardized American Society for Testing and Materials (ASTM) guide for the conduct of FETAX has been published, along with a companion atlas that helps in embryo staging and in identifying malformations. As part of the ASTM process, an interlaboratory validation study was undertaken to evaluate the repeatability and reliability of FETAX and to evaluate the potential teratogenic hazard of 12 compounds. Three different laboratories participated in the study. All three participating laboratories had extensive experience with the assay. FETAX intralaboratory and interlaboratory variability, as judged by coefficients of variation, were very low. Potential teratogenic hazard was evaluated using two major criteria from FETAX experiments employing metabolic activation systems (MAS). These were the teratogenic index TI (TI = 96-h lc(50)/96-h ec(50) (malformation)) and the minimum concentration that inhibits growth (MCIG). A compound was considered teratogenic by this criterion when the MCIG was significantly different from controls at concentrations below the 30% level of the MAS 96-h lc(50). Based on the results of this and other studies, a decision table was constructed in order to evaluate additional studies. Severity of malformations caused, especially near the MAS 96-h ec(50) (malformation), were also evaluated. Four compounds were non-teratogenic but two compounds were clearly teratogenic. The remaining six compounds were ranked as equivocal teratogens. The results were discussed in light of the difficulty of producing an adequate decision table. FETAX proved to yield repeatable and reliable data as long as care was taken during range-finding and technicians were adequately trained. The MAS was essential in using FETAX to predict developmental hazard in mammals, and still requires further development.

Abstract : Several species of teleosts, including medaka (Oryzias latipes), have been studied with respect to neoplastic responses to known carcinogens. Little information, however, is available concerning the responses of the immune system to environmental chemicals in the medaka. We have initiated studies to characterize immune organs and function in medaka, with the goal of using the medaka as a predictive model of immnunotoxicity in vertebrates. Development of a teleost model for immunotoxicity is part of an integrated biological approach for assessment of the hazard resulting from exposure to complex chemical contaminants in the environment. Medaka immune organs essential for leukopoiesis/erythropoiesis as well as cell morphology were examined. Cells isolated from the anterior kidney, spleen and whole blood were characterized with respect to nonspecific esterase activity, myeloperoxidase activity and acid phosphatase activity. in vitro phagocytic cell function following in vitro activation was assessed in primary cultures of pronephros adherent cells. Data collected to date characterizing the endogenous bacterial flora of medaka cultured in our laboratory is also presented.

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was used to assess the teratogenic potential of four solvents. Embryos of the South African clawed frog, Xenopus laevis, were exposed for 96 h to ethanol, dimethyl sulfoxide (DMSO), formamide or glycerol formal. Exposure groups were maintained using a static renewal system in which the exposure media were changed at 24-h intervals. Survival was monitored at 24-h intervals. Length, as an indicator of growth effects, and developmental malformations were determined at the end of the assay (96 h). Using this information, the 96-h LC50, the 96-h EC50 (Malformation), and the no observable effect levels (NOELs) for mortality, malformation and length were determined for each solvent. The teratogenic index [TI = 96-h LC50/96-h EC50 (Malformation)] also was calculated for each of the solvents. DMSO appeared to be the least toxic or teratogenic solvent examined, with a pooled LC50 of 1.92%, a pooled EC50 (Malformation) of 1.57% and TI values of 1.20 and 1.24 in replicate trials. Formamide appeared to be the most toxic solvent, with a pooled LC50 of 1.04%. Data trends suggested that ethanol was the most teratogenic solvent tested, with a pooled EC50 (Malformation) of 1.04% and TI values of 1.42 and 1.50. The results obtained in the present work for ethanol and DMSO were compared to previously published FETAX results for these two solvents. The present results are in close agreement with these results from other laboratories, thus providing further evidence supporting the interlaboratory reproducibility of FETAX results.

Growth and ChangeVolume 16, Issue 2 p. 26-33 An Interindustry Approach to Financing Small Port Development and Maintenance ROBERT A. FINCH, ROBERT A. FINCH Robert A. Finch is an economist with the U.S. Corps of Engineers in Wilmington, North CarolinaSearch for more papers by this authorMARK S. HENRY, MARK S. HENRY Mark S. Henry is a professor of agricultural economics at Clemson University.Search for more papers by this author ROBERT A. FINCH, ROBERT A. FINCH Robert A. Finch is an economist with the U.S. Corps of Engineers in Wilmington, North CarolinaSearch for more papers by this authorMARK S. HENRY, MARK S. HENRY Mark S. Henry is a professor of agricultural economics at Clemson University.Search for more papers by this author First published: April 1985 https://doi.org/10.1111/j.1468-2257.1985.tb01046.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume16, Issue2April 1985Pages 26-33 RelatedInformation

The distribution of concanavalin A (con A) binding sites on chick wing-bud mesoderm cells from two different developmental stages and from different regions of the same embyronic limbs was examined. Fluorescein isothiocyanate (FITC)-labeled con A binding sites on mesoderm cells from whole stage 19–20 and the distal third of stage 25 limbs redistributed from a random array over the cell surface to form clusters or caps of binding sites. In contrast, a significantly smaller percentage of developmentally “older” mesoderm cells from the proximal third of stage 25 limbs underwent redistribution of bound con A from its normal random arrangement. In order to further examine the basis for the decrease in con A redistribution in the stage 25 proximal cells, this segment was subdivided into a core (presumptive chondrogenic) and peripheral (presumptive myogenic) region. The results of con A redistribution studies utilizing these two cell populations indicate that a significantly smaller percentage of the core cells are capable of forming clusters or caps. Likewise, in agglutination studies, core cells proved significantly less susceptible to con A-induced agglutination. These findings indicates that the differences in agglutination and con A redistribution, seen when comparing the proximal cells with the other cell populations, is attributable to a subpopulation of the proximal cells, specifically, those located in the chondrogenic core region. Since the proximal core and peripheral cells are of the same developmental age, these findings suggest that developmental age is not solely responsible for the differences reported here. Thus, prospective phenotype appears also to be involved in the decreased agglutination and a con A redistribution of the chondrogenic core cells.

The observation that cells often respond to carcinogens by nuclear enlargement has suggested that this property might be useful to develop a short-term screening test for such compounds. Previous methods for detecting nuclear size increases have used an image analyzer system to detect nuclear changes in individual cells. This paper details a more rapid method for obtaining nuclei by use of a stromalyzing procedure following by analysis of nuclear volumes, using a Coulter Counter Channelyzer. This new and simplified nuclear sizing method should facilitate the use of the assay as a possible carcinogenesis screen by permitting rapid and efficient testing of large numbers of compounds.

The in vitro chondrogenic patterns expressed by mesoderm cells obtained from the proximal, middle, and distal thirds of stage 25 chick embryo wing buds were studied. Region-dependent differences were observed in the patterns of Toluidine Blue-staining extracellular matrix produced. These differences were subsequently shown to be highly reproducible. Chondrogenesis in cultures containing cells from the proximal third of the limb is restricted primarily to the center of the dish and takes the form of small nodules by day 4. These nodules coalesce into a large colony by day 6. In cultures containing similar numbers of cells from the distal third of the limb, aggregates of cells surrounded by metachromatic extracellular matrix materials are detectable somewhat earlier than in those containing cells from the proximal region. A centrally located concentration of matrix material is found in these distal cell cultures, however, a large number of nodules are also scattered throughout the monolayer. The difference between the chondrogenic patterns seen in the proximal and distal cell cultures becomes more pronounced by day 6 of culture. This is due primarily to the absence of demonstrable cartilaginous matrix in the peripheral areas of the proximal cell cultures. Cultures composed of cells derived from the middle third of stage 25 wings exhibit patterns of Chondrogenesis intermediate in appearance between those containing cells from the other two regions. It is unlikely that these readily repeatable differences in chondrogenic patterns can be explained solely by convection currents in the media caused by the seeding and handling of the cultures prior to attachment of the cells. The different chondrogenic patterns might therefore be suggestive of region-dependent differences in the cell surface characteristics of limb mesoderm cells.

The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20--21, 23--24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 microgram/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20--21, 23--24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20--21 or 23--24 embryos. It was determined that culture medium composed of Eagle's MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Ham's F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.

Cytochalasin B (CB) has been shown to have many biological effects on cultured cells. We report that an initial 48-hr treatment of freshly plated chick embryo limb mesoderm cells with CB irreversibly inhibits chondrogenesis. A slight inhibition in the amount of matrix is seen when limb cells are allowed to grow in culture for 24 hr prior to treatment for the second 24 hr of culture. If the cells are allowed to plate-out and grow for 48 hr or longer prior to being treated with CB for 24 hr, the amount of matrix produced is essentially the same as that seen in the controls. However, if the initial 48-hr culture period is followed by a 48- or 72-hr treatment, chondrogenesis is reduced, but not to the same extent as that seen in cultures treated for the first 48 or 72 hr. The irreversible inhibition of chrondrogenesis does not appear to be due to irreversible inhibition of protein synthesis or hexose uptake because, although these are reduced during treatment, they return to control levels within 48 hr following the removal of the drug. We cannot mimic the effect of CB treatment using glucose-deficient medium, thereby eliminating the possiblity that a critical glucose level is necessary to permit chondrogenesis. Multinucleation of limb cells treated with CB is reversed within 4 to 7 days following the removal of the drug. Therefore multinucleation alone is probably not responsible for the CB effect on chondrogenesis. However, other subtle permanent changes may occur during the period of multinucleation which result in the irreversible inhibition of chondrogenesis.

MORPHOGENETIC potential of embryonic chick limb mesoderm cells declines with time beginning at approximately stage 24 of development1. This decline occurs in a proximo-distal direction2. Accompanying the loss of morphogenetic potential there is an age-dependent decline in the ability of limb and axial mesoderm to sort from one another in a randomised mixture of cells1,3,4. Due to the age-dependent decline in both morphogenetic potential and the sorting ability of the limb mesoderm cells, it has been suggested that the decline in morphogenetic potential may be related to changes occurring at the cell surface5. Cell surface carbohydrate moieties have been postulated to play an integral part in cellular interactions6. Because of their high degree of carbohydrate-binding specificity7, plant lectins, such as concanavalin A (con A), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA), have been used to monitor cell surface changes which occur during development7–13. These changes manifest themselves as differences in lectin-induced agglutinability of cells, and changes in number and distribution of lectin binding sites on the cell surface. This study was designed to determine if there were cell surface changes accompanying the decline in morphogenetic potential of the limb mesoderm cells, which could be detected by the use of plant lectin probes. We report here that there is a slight increase in RCA-induced agglutination of chick wing-bud mesoderm cells with increasing developmental age. There is also a significant age-dependent, proximo-distal decline in the susceptibility of chick wing-bud mesoderm cells to con A-induced agglutination between stages 19 and 26 of development. As such, there is a spatial as well as a temporal correlation between the decline of con A agglutinability and the decline of the morphogenetic potential of the chick limb-bud mesoderm cells.

In this study dissociated late stage 20 (stage 20+) chick leg-bud mesoderm cells cultured in cycloheximide (1 μg/ml) were tested to determine the retention of their ability to undergo recognizable limb morphogenesis, according to the technique described by Zwilling (1964). The cycloheximide treatment retards the loss of this morphogenetic property compared with the normal loss that occurs in cultures of untreated cells (Finch and Zwilling, 1971). The exposure to cycloheximide extends the period during which this morphogenetic property can be detected for approximately 15–20 hr. Incorporation experiments show that cycloheximide at 1 μg/ml rapidly inhibits the synthesis of protein. RNA, and DNA in cells exposed to the antibiotic. This inhibitory effect is completely reversible in a relatively short time after removal of the cycloheximide.

Stability of morphogenetic properties of leg bud cells (stage 20+) after two culture procedures was tested with a technique described by Zwilling ('64). This technique involves the placing of the cells to be tested into ectodermal jackets obtained from stage 20–21 limb buds and then grafting the composites either to the dorsum of the wing bud or over the somites of a host embryo. There is a decline in the limb properties between 12 and 18 hours of either shaking flask or plate culture. This decline parallels that observed in the limb bud in vivo. The distal area of the wing dorsum as a graft site partially overcomes the decline of the limb properties. Plating at high cell density retards the decline in morphogenetic properties. It is concluded that the culture conditions used in the present experiments have little, if any, effect on the stability of the morphogenetic properties of leg bud cells. Loss of these properties appears to be time dependent. This situation has possible implications for future in vitro studies of the molecular basis for the properties of the morphogenetic phase of chick limb development.