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D M Kochhar

Publications and source records attributed to D M Kochhar.

At least 55 records · Page 3Linked to original sources
PubMed1984

Retinoic acid enhances the displacement of newly synthesized hyaluronate from cell layer to culture medium during early phases of chondrogenesis.

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid (1 microgram/ml or 3.3 X 10(-6) M). Since in control medium overt chondrogenesis begins on day 3, retinoic acid was introduced on day 2 so that the relationship between initial biosynthetic changes and inhibition of chondrogenesis could be studied. During the first 24 h of exposure the treated cells remained viable but suffered 10% inhibition in growth and synthesized [3H]glucosamine-labeled glycosaminoglycan at a level 24% below untreated cells. The amount of labeled hyaluronic acid released into the culture medium by the treated cells was, however, 2-fold greater, on a per cell basis, than that in the untreated cultures. It is suggested that the displacement of hyaluronate may play a role in the disruption of mesenchymal cell differentiation and of limb morphogenesis as observed in other systems.

Animals↗
PubMed1983

A selection of candidate compounds for in vitro teratogenesis test validation.

The Consensus Workshop on In Vitro Teratogenesis Testing recommended that test validation be facilitated by a listing of agents with defined teratogenicity; subsequently, a panel was convened to review and select such agents. This communication established a list of 47 compounds or conditions which demonstrate a wide range of teratogenicity in vivo. The agents were chosen primarily on the strength of the literature base denoting their in vivo effects. The tables note a number of general biological and toxicological characteristics for each agent, and the details of representative in vivo teratology studies are summarized and referenced. This list is intended to serve as a base for in vitro teratogenesis test validation and should prove useful in developing and identifying those systems which will contribute to a more effective testing program.

Abnormalities, Drug-Induced↗
PubMed1982

Embryonic limb bud organ culture in assessment of teratogenicity of environmental agents.

The limb bud organ culture system offers a variety of endpoints which may be monitored in the screening process. These are: cell proliferation, differential growth, morphogenetic cell death, size and shape of limb parts, chondrogenesis, collagen or proteoglycan biosynthesis, etc. Essentials of the system including various parameters of normal limb bud development in vitro are described. These parameters serve to gauge the effects of test chemicals with unknown hazard potential. Validation has been carried out only to a limited extent. Further, it needs to be combined with an efficient drug metabolizing preparation before it can achieve its full potential as a short-term screening system.

Acetates↗
PubMed1981

Limb development in mouse embryos. III. Cellular events underlying the determination of altered skeletal patterns following treatment with 5'fluoro-2-deoxyuridine.

A potent inhibitor of thymidylate synthetase, 5-Fluorodeoxyuridine (FUdR), produced dose- and stage-dependent skeletal defects in embryos of mice injected during early development (10th--13th day). The dose of 25 mg/kg was teratogenic producing few resorptions and little reduction in fetal weight. Hindlimbs were much more sensitive to the drug than were the forelimbs and showed dose-dependent long bone reductions. Specific long bone reductions occurred after treatment on the 11th or 11.5 day and were accompanied by preaxial polydactyly; treatment on the 12th or 12.5 day produced high incidence of ectrodactyly but no long bone reductions. Limbs were insensitive on the 13th day of development and beyond. Limb buds in organ culture demonstrated a direct action of FUdR on undifferentiated mesenchymal cells, incurring what has been previously termed "thymineless" cell death. Dose-dependent reduction of viable cell number led to a reduction in chondrogenic expression in vitro--more so in the hindlimb than the forelimb. Similar occurrence of cell death was also noted after in utero exposure, and here, it was followed by a process of recovery. The recovery phase was characterized by the formation of a distal synchronized cell population derived from undamaged subridge cells leaving damaged cells at the proximal extent of the subridge zone. Limb segments maintained their temporal sequence of differentiation, and their regulatory capability was related to the time allotted for repopulation of the subridge zone. We propose that a faster growth during early development of the elements of the hindlimb versus analogous ones of the forelimb allows the former less time for regulation--and hence, higher sensitivity to antiproliferative agents such as FUdR.

Animals↗
PubMed1981

The role of morphogenetic cell death during abnormal limb-bud outgrowth in mice heterozygous for the dominant mutation Hemimelia-extra toe (Hmx).

A dominant mutation in the mouse, Hemimelia-extra toe (Hmx), induces congenital limb malformations in heterozygotes. Typical expression includes axial shortening of the radius, tibia and talus ('hemimelia'), with supernumerary metacarpals, metatarsals, and digits ('polydactyly'). Pathogenesis was investigated during developmental stages 16 through 22 (11th through 15th days of gestation). Full expression was apparent during stage 20 when the limb pattern was comprised of pre-cartilaginous anlagen. Formation of a pre-axial protrusion on the autopod during stage 17 or 18 was the earliest gross abnormality, and foreshadowed the development of supernumerary digits. Microscopically, there was an alteration in the pattern of physiologic cellular degeneration (PCD) programmed to occur within the zeugopod and autopod. The 'opaque patch' (mesodermal necrotic zone normally occurring between tibial and fibular anlagen) was overextended pre-axially causing resorption of the tibial precartilage. Additionally, PCD normally occurring within the basal cell layer of the apical ectodermal ridge (AER) and the 'foyer primaire préaxial' was not expressed in the mutant autopod. This occurred in association with outgrowth of the protrusion. The pre-axial portion of the AER remained in an abnormally thickened, viable, proliferative state, and did not undergo scheduled degression. This may have been the basis for prolonged induction of pre-axial outgrowth. Paucity of mesenchymal cell filopodial processes extended along the basal lamina, as well as a rarefaction of the filamentous material normally associated with the mesodermal face of the basal lamina, was detected at the pre-axial AER-mesenchymal interface on stage 18. A potential involvement of epithelial-mesenchymal interactions in the induction of epithelial PCD is discussed.

Animals↗
PubMed1980

In vitro testing of teratogenic agents using mammalian embryos.

The need for efficient methods to screen new chemicals, drugs, and environmental pollutants for their teratogenic activity is obvious. The method currently available, ie, pregnant-animal testing, is of considerable value but there are certain drawbacks which prevent reliance on this method alone as the predictive device. Nutritional state of the dam, variability in the developmental age of embryos from litter to litter and even within the same litter, metabolic differences between species, placental function, and a host of other factors must be taken into account before data obtained from animal testing can be logically extrapolated to human situation. Many of these variables are either eliminated altogether or at least can be controlled by the use of tissue culture techniques. After surveying a variety of in vitro systems, it is our opinion that organ culture, whole embryo culture, and a combination of the two offer at present the best potential for screening of suspected teratogens. These culture techniques provide a much better simulation of in vivo situations than isolated cells grown as monolayers. Among other advantages, these procedures allow one to exercise control over the effective concentration of the suspected teratogen to which an embryo is exposed and also the duration of this exposure. Since maternal metabolism or modification of the drug is routinely eliminated in these experiments, there is a need for exploring the use of drug-metabolizing preparations as additives to the culture medium. The choice of limb bud in the screening system is promising since during its development, the limb progresses through a succession of embryonic processes that are generally relevant to other organ systems as well. Hence, such a screening system may not only predict teratogenicity but also provide insight into the mechanisms by which a test chemical is teratogenic.

Abnormalities, Drug-Induced↗
PubMed1980

Production of congenital limb defects with retinoic acid: phenomenological evidence of progressive differentiation during limb morphogenesis.

Maternal administration of a single dose of retinoic acid (vitamin A acid, 100 mg/kg) on either the 11th, 11 1/2, 12th, 12 1/2, 13th or 13 1/2 day of gestation produced phocomelia or partial phocomelia in ICR/DUB fetuses. The results depended upon the time of treatment and two gradients of effect were produced: 1) cranio-caudal gradient, since forelimb defects resulted from between days 11 and 13, while similar hindlimb abnormalities were produced by administration of retinoic acid 12 to 24 hours later: 2) proximo-distal gradient, due to the heterogenous sensitivity among individual bones of the limb. In the forelimb, early treatment (11th day) produced humero-unlar defects and later treatment (12th day) ulnoradial defects. A similar proximo-distal gradient was observed in the hindlimb. The use of teratological studies as a tool to assist morphogenetic investigation is discussed.

Abnormalities, Drug-Induced↗
PubMed1980

Vitamin A alters the internal viscosity of fragments of limb-bud mesenchyme.

Two techniques were used to examine the effect of vitamin A compounds (vitamin A acid = retinoic acid and vitamin A acetate) upon the relative strengths of adhesion among mouse limb-bud mesenchymal cells. Treatment with retinoic acid in vivo and with vitamin A acetate in vitro reduced the rate at which the fragments of mesenchyme rounded-up when cultured on a non-adhesive substratum, but these compounds did not alter the behavior of tissues tested in fragment-fusion experiments. These conflicting results indicate that the two tests measure different activities of cells and suggest that treatment with vitamin A alters the property(ies) of cells which regulate the internal viscosity of tissues.

Animals↗
PubMed1978

Limb development in mouse embryos. II. Reduction defects, cytotoxicity and inhibition of DNA synthesis produced by cytosine arabinoside.

Various morphological and biochemical parameters were used to study the mode of interference by cytosine arabinoside (Ara-C) in critical phases of embryonic limb development. Inhibition of embryonic DNA synthesis occurred immediately after injection of Ara-C into the mother. The inhibition was dose-dependent and was substantial even after the nonteratogenic dose (2 mg/kg) of Arc-C. The pattern of limb bone deficiencies in Ara-C treated fetuses was specific for each developmental stage at which the treatment was given; the site of affect moved distalwards along the limb as the development advanced. The teratogenic dose was cytotoxic to mesenchymal cells with a high proliferation rate but did not affect others such as the future cartilage cells in which the rate of proliferation was lower. The existence of this differential susceptibility at each stage of development, together with information about the pattern of bone defects at the same stage, permitted us not only to define with some precision the cellular basis of origin of limb defects but also to infer the relative level of cell differentiation pertaining to each successive stage. Deoxycytidine, if injected simultaneously with and at doses eight times larger than Ara-C, afforded virtually complete protection against teratogenic effects. Deoxycytidine also prevented cell death in the limbs of Ara-C treated embryos. However, a dramatic increase in the frequency of polydactyly was found in the protected fetuses. The fact that the frequency of ectrodactyly in the protected fetuses decreased in inverse proportion to the frequency of polydactyly strengthened the notion that there may be a common cellular basis underlying these two types of digital defects. Striking changes were found in the structure of AER at 24 hours after Ara-C treatment; it was abnormally thickened into a gland-like structure and its inner edge facing the mesenchyme thickened into a gland-like structure and its inner edge facing the mesenchyme was thrown into several folds. This may constitute a response to impairment in the underlying mesenchyme with which AFR has long been considered to have an interdependent relationship.

Animals↗
PubMed1976

Transplacental passage of label after administration of (3H) retinoic acid (vitamin A acid) to pregnant mice.

To determine whether there is transplacental passage of retinoic acid (vitamin A acid) or its metabolites levels of radioactivity were measured in embryos after administration to 12th-day pregnant ICR/DUB mice of nonteratogenic or teratogenic quantities of (3H)retinoic acid. After the former treatment radioactivity was demonstrable in embryos, placentas, and visceral yolk sacs (VYS) at 1 h after treatment. Expressed in terms of radioactivity per unit protein content, embryos at 1 h had about one-third as much radioactivity as maternal livers. The placentas had maximum radioactivity at 2 h, whereas embryos and VYS accumulated maximum levels by 6 h after treatment. At 6 h the level in embryos was twice that in placentas. After teratogenic treatment placentas and embryos attained peak levels of radioactivity at 6 and 12 h later, respectively. Again, embryos at peak level contained 2-3 times as much radioactivity as placentas or VYS. The amount of retinoic acid, metabolites, or both, in embryos after the teratogenic treatment, calculated on the basis of specific activity of the injected label, was 26 times greater than after nonteratogenic treatment; the amount in maternal liver was 75 times greater. The results support previous evidence that the teratogenic effects of retinoic acid are associated with raised levels of retinoic acid, its metabolites, or both, in embryos.

Animals↗