Changes in hepatocyte ploidy in response to chromium, analyzed by computer-assisted microscopy.

BDF1 mice were given single injections of sodium dichromate (25 mg/kg) on an acute (6 hr to 7 days) or intermediate (2-4 weeks) basis, or multiple injections (12.5 mg/kg) on a chronic (4.5 months) basis. Observed hepatic changes included programmed cell death (apoptosis) in the periportal region with acute exposure and fusion of liver lobes with chronic exposure. Response to chromate exposure was measured by change in hepatocyte nuclear ploidy state (e.g., the proportion of diploid, tetraploid, and octaploid nuclei) based on computer-assisted imaging from histological sections. The computer-assisted imaging system used in this study was superior to traditional methods because it (1) allows rapid ploidy determinations from histological material and (2) can be used to collect regional information. Regional differences in ploidy were seen to occur in a consistent fashion among both control and treated animals. Nuclei adjacent to the portal triad had the lowest ploidy value (highest proportion of diploid nuclei), an intermediate value was found adjacent to the central vein, and the highest ploidy was found in the midzone. These three ploidy-based zones roughly correspond to the three functional zones of A. M. Rappaport (1973, Microvasc. Res. 6, 212-228) and W. H. Lamers et al. W. H. Lamers, A. Hilberts, E. Furt, J. Smith, G. N. Jonges, J. F. Van Noorden, J. W. G. Janzen, R. Charles, and A. F. M. Moorman, 1989, Hepatology, 10, 72-76. Temporal changes in ploidy were seen among control animals (all zones), with young animals (56 days) displaying relatively low ploidy values compared to older animals (184 days). Chromate exposure caused increased ploidy (all zones) among animals treated on an acute basis (the youngest animals). Chromate had no apparent effect on ploidy among animals treated for longer periods of time, probably because of age-related factors.

Computer-based image analysis of cartilage differentiation in embryonic limb bud micromass cultures.

A computer imaging system was used to analyse the effects of benzamide (BAM), sodium butyrate and dimethyl sulphoxide (DMSO) on chondrogenesis in culture. Embryonic chick limb bud cells were used as a source of undifferentiated mesenchyme, and cultures were prepared using a micromass culture technique. The degree of chondrogenesis is directly related to the amount of Alcian blue staining. Standard assays include either manual tally of individual cartilage nodules, which is both tedious and time-consuming, or extracting bound dye for spectrophotometric analysis, which obscures individual variations because it requires pooled samples. In the present study, low-magnification images of individual micromass colonies were converted into derived images based on their percentage transmittance relative to the background. These derived images were analysed for relative degree of chondrogenesis, using area and integrated optical density (IOD) as descriptors. While both types of data were useful for ranking the degree of chondrogenesis in culture, IOD was the preferred descriptor because of its accuracy over a wide range of threshold values. The area and IOD data both demonstrate that BAM produces marked enhancement of chondrogenesis in culture, sodium butyrate causes marked inhibition and DMSO produces mixed results (enhancement at the high dose, inhibition at the low dose). While the present study demonstrates the usefulness of computer-aided microscopy for analysis of low-magnification images, the same descriptors (area and IOD) should be useful in quantifying data from a variety of objects (cells, nuclei, etc.) which can be stained in a selected, quantitative fashion.

The effects of gamma radiation on chondrogenic development in vitro.

Gamma radiation (0.9-8.0 Gy) was used as a perturbing agent to study factors influencing in vitro chondrogenesis of embryonic chick limb bud cell culture. Chondrogenesis was measured using a number of criteria, including (1) cartilage nodule production, (2) spectrophotometric determination of the amount of bound Alcian blue dye, and (3) computer-assisted analysis of the spatial distribution (area) and density of Alcian blue present in individual micromass colonies. Gamma radiation inhibited both cell proliferation and chondrogenesis in a dose- and time-dependent fashion. Administration of benzamide caused a significant increase in cell proliferation at 0.9 and 2.7 Gy, and in chondrogenesis at all doses. Cartilage nodule production was affected during the first 2 days (prior to 48 h) of culture only, suggesting that chondrocytic commitment occurs during this period. Cultures irradiated at 48 and 72 h produced the same number of nodules as controls, but bound significantly less dye, presumably because of decreased cell numbers and/or cell synthesis products. Computer analysis of micromass colonies provided data similar to those collected spectrophotometrically, but displayed the advantages of (1) increased sensitivity to individual variations, (2) the ability to collect data sets without having to pool three or more colonies, and (3) long-term storage of raw images for later analysis.

Effect of phorbol and bryostatin I on chondrogenic expression of chick limb bud, in vitro.

The ability of phorbol esters to promote tumor formation and alter cell differentiation has been attributed to its action on a number of critical cellular functions, in particular, on protein phosphorylation, through the activation of protein C kinase. The present paper describes the effects of PMA (phorbol 12-myristate 13 acetate) on in vitro chondrogenesis in non-passaged, embryonic limb bud cells, relative to the effects of Bryostatin I. This compound also activates C kinase and binds competitively to the phorbol ester receptor, yet does not affect cell differentiation. Levels of PMA as low as 10(-7) M markedly reduced cartilage formation in 4-day cultures, as indicated by nodule count and Alcian blue staining for chondroitin sulfate. Coadministration of Bryostatin I at equimolar concentration prevented the PMA inhibitory effect on chondrocytic expression. This confirms other findings that phorbol activation of C kinase cannot exclusively account for the activity of phorbol on cell expression, i.e., that other pathway(s) must also be involved. Altering the time of PMA exposure demonstrated that PMA inhibited chondrocyte phenotypic expression, rather than cell commitment: early (0-48 h) exposure to PMA (during chondrocytic commitment in vitro) had little inhibitory effect on the staining index, whereas, exposure from 49-96 h (presumably post-commitment) and 0-96 h had moderate and strong inhibitory effects, respectively, on cartilage synthesis. Further research on the phorbol/Bryostatin I interaction should add to our knowledge of the control processes involved in tumor promotion and cell differentiation.

Distribution and HPLC study of chromium-51 binding sites in Chinese hamster ovary cells.

Chinese hamster ovary cells were cultured with chromium-51 chromate to study the site of chromium interaction with cell biomolecules. After incubation, cells were homogenized and separated into nuclear, mitochondrial, microsomal, and cytosolic fractions. Greater than 75% of the radioactivity was found in the cytosolic fraction. The supernatant from the centrifuged cell homogenate, which contained greater than 90% of the chromium radioactivity, was subjected to chromatographic investigation. The combination of anion exchange and ion-pair high-performance liquid chromatography (HPLC) showed that at least three different molecular species interact with chromate or its reduced derivative, Cr(III). These species are glutathione, the nucleotides cytosine triphosphate, uridine triphosphate, guanine triphosphate, adenosine triphosphate, and adenosine diphosphate, plus an as yet unknown species of protein or peptide. Preliminary data for the specific activity of nucleoside triphosphates range from 6000 to 18,000 cpm/micrograms ribonucleoside triphosphate. The glutathione accounted for 50% of the observed radioactivity, the nucleotides for 30%, and the metalloprotein accounted for the remainder.

Chromium effects on chondrocytic differentiation in vitro.

A tissue culture study was conducted on the effects of chromium on chondrocytic differentiation. Mesenchymal cells from stage 22-24 chick limb buds were dispersed and cultured as micromasses, where they differentiated into chondrocytes. Addition of chromium(VI) to the cultures indicated that the production of proteoglycans (as detected by Alcian blue staining) was more sensitive to chromium's effects than was cell proliferation. Whereas Alcian blue nodule formation was inhibited by 1 microM Cr(VI), cell proliferation (as detected by cell counts) was not. Chromium (VI) was added to cultures at daily intervals, and these studies indicated that the interval of d 1-2 was the most sensitive period. ADP-ribose transferase activity in these cultures was measured; the pattern of enzyme activity in control cultures was high 1 and 24 h after the start of culture, decreased abruptly between 24 and 48 h, and then decreased more gradually. In the presence of Cr(VI), elevated ADP-ribose transferase levels were maintained throughout the culture period. We suggest that, in presence of 1.0 microM chromium(VI) or higher concentrations, the balance of events favors nucleolytic action rather than repair of damage.

Benzamide on chondrocytic differentiation in chick limb bud cell culture.

Benzamide, an inhibitor of (ADP-ribose) transferase, augmented chondrocytic differentiation of chick limb bud mesenchymal cells in micromass cultures; the incorporation of 35SO4(2-) into the trichloroacetic-acid-insoluble constituents of cell masses as well as the formation of cartilage nodules (Nishio, Nakanishi, Doull & Uyeki, 1983) occurred about 24 h earlier than in untreated cultures and continued to be enhanced in benzamide-treated cultures of stage 23- to 24-chick limb bud cells. Benzamide also significantly increased cell proliferation. However, benzamide did not affect DNA and RNA syntheses except for one period: 24 to 30 h after the start of culture, RNA synthesis was stimulated. From 48 h of culture, (ADP-ribose) transferase activity decreased daily in untreated cultures, whereas benzamide treatment diminished (ADP-ribose) transferase activity 24 h earlier. On the other hand, intracellular NAD levels increased daily in untreated cultures, and benzamide significantly increased the NAD levels above untreated cultures. ATP levels did not differ significantly during the culture period, and benzamide did not affect ATP levels.

Inhibition of DNA synthesis by chromium compounds.

Cr(VI) irreversibly inhibited DNA synthesis in cultured mouse L cells to 50% of controls at 10 microM; 3.3 mM Cr(III) did not. At 0.3 mM, Cr(III) and Cr(VI) inhibited DNA synthesis in permeabilized L cells to 50% of control values. Cr(III) was a stronger inhibitor of DNA synthesis in the DNA-Escherichia coli DNA polymerase I system than was Cr(VI). The inhibitory effect of Cr(VI) depended on the ratio of Cr/DNA and Cr/enzyme; on the other hand, the increase in the concentration of DNA polymerase did not affect the inhibition of Cr(III), Cr(III), below the inhibitory concentration, produced an increase in the incorporation of [3H]dTMP into DNA; this was not observed with Cr(VI).

Effect of benzamide on cell growth, NAD and ATP levels in cultured chick limb bud cells.

Benzamide, an inhibitor of poly (ADP-ribose) synthetase, which augments chondrocytic differentiation, significantly decreased poly (ADP-ribose) synthetase activity in cultured chick limb bud cells. Benzamide significantly increased cell proliferation, but did not affect DNA synthesis per cultured cell. Furthermore, benzamide significantly increased NAD levels and synthesis, while it did not affect ATP levels and synthesis except for ATP synthesis on day 1. Thus, we suggest that benzamide augments both cell proliferation and NAD synthesis during chondrocytic differentiation.

Induction of DNA strand breaks and chromosome abnormalities by an imide derivative of 3-nitro-1,8-naphthalic acid (mitonafide) in Chinese hamster ovary cells.

Mitonafide (5-nitro-2-(2-dimethylaminoethyl)-benzo- [de]isoquinoline-1,3-dione hydrochloride), a new candidate as an anticancer or antiviral agent, inhibited the incorporation of DNA precursors into the acid-insoluble fractions of cultured Chinese hamster ovary (CHO) cells and also into CHO cells made permeable (i.e., "permeabilized") that were supplemented with ATP and deoxynucleoside triphosphates. Mitonafide enhanced the degradation of previously incorporated [3H]thymidine and increased the amount of DNA recovered in fractions containing single-stranded DNA after alkaline denaturation and hydroxyapatite chromatography. At concentrations of 0.01 and 1.0 microM, respectively, mitonafide increased the frequency of sister chromatid exchanges and chromosome aberrations in CHO cells. The inhibition of DNA synthesis in a permeabilized cell system suggested that mitonafide acted on the DNA-synthesizing apparatus without requiring metabolic conversion and interfered with DNA synthesis independent of the cellular metabolism of DNA precursors. Mitonafide induced DNA strand breaks and chromosome abnormalities in cultured cells.

Cellular uptake and inhibition of DNA synthesis by dihydroxyanthraquinone and two analogues.

Three analogues of aminoalkylamino-substituted anthraquinone derivatives, namely, 1,4-dihydroxy-5,8-bis(((2-[(2-hydroxyethyl)amino]ethyl)amino))-9,10-anthracenedione (DHAQ), 1-hydroxy-5,8-bis(((2-[(2-hydroxyethyl)amino]ethyl)amino))-9,10-anthracenedione (HAQ), and 1,4-bis(((2-[(2-hydroxyethyl)amino]ethyl)amino))-9,10-anthracenedione (AQ), were chosen with respect to the number of hydroxyl groups on the aromatic ring. DHAQ showed about 100 times more potent antiproliferative activity on cultured mouse L-cells than did AQ; HAW showed intermediate activity. This antiproliferative activity was correlated with their inhibitory effect on DNA synthesis in culture. When their inhibitory effect on DNA synthesis was conducted in a permeabilized L-cell assay, all compounds were inhibitory; the order of potency was DHAQ greater than HAQ greater than AQ. The same order of potency was also observed in calf thymus DNA and Escherichia coli DNA polymerase I system. Their inhibitory effect in the latter system was correlated with the drug:DNA molar ratio, and not with drug:enzyme ratio. Comparative uptake of the drugs by intact L-cells showed the highest uptake of DHAQ followed by those of HAQ and AQ. The large differences in their uptake by intact cells became minimal when cells were rendered permeable to exogenous materials or when nuclei were used. Hence, these studies revealed that the hydroxyl group on the aromatic ring of the compounds influenced their biological activity not only by potentiating drug-target interaction but also by drug uptake into cells.

Enhanced chondrocytic differentiation in chick limb bud cell cultures by inhibitors of poly(ADP-ribose) synthetase.

Inhibitors of poly(ADP-ribose) synthetase, namely nicotinamide, benzamide, m-methoxybenzamide and 3-aminobenzamide, augmented chondrocytic differentiation chick embryo limb bud mesenchymal cells, in culture. These inhibitors stimulated early appearance and massive formation of cartilage nodules in micromass cultures stage 23-24 chick embryos. They also induced nodule formation in micromass and cartilage colonies at micromass plating densities from stage 18-19 embryo Benzamide, however, did not prevent differentiated chondrocytes from undergoing a pleiotypic change in cell type. These results are compatible with the putative regulatory function of poly(ADP-ribose) on cell differentiation.

Teratogenic effects of cholinergic insecticides in chick embryos. III. Development of cartilage and bone.

Teratogenic effects of two organophosphate insecticides, diazinon and dicrotophos, were investigated in regard to skeletal development, particularly of the extremities and vertebrae. Cartilage and calcified bone were examined with alcian blue and alizarin red S staining techniques, respectively, in chick embryos of d 5 to 17 of incubation. The age-related development of both cartilaginous and ossified portions of the hind leg was measured in control and insecticide-treated groups. Diazinon and dicrotophos (200 micrograms/egg), injected on d 3, inhibited growth of the following skeletal elements: femur, tibia, metatarsi and digits of the leg. The inhibition was noticeable from the 9th d of incubation. The greatest reduction of the skeletal length was observed in tibia and metatarsi, and was characterized by angulations toward the dorsal side. Percent of growth inhibition of the calcified region in the legs was similar to that of the entire length of each skeletal element, but there was no difference between control and insecticide-treated groups on the time-related appearance of cartilaginous or calcified long bones, digits, and phalanges of legs. In the cervical region of embryos treated with the insecticides, unique deformities such as an "undulating" notochord and fused cervical rings were seen at an early stage (d 6). We suggest that the organophosphate-induced malformations in legs are mainly due to growth retardation of later stages of development of each skeletal element. On the other hand, the neck deformities result from a profound alteration of differentiation at early stages of development.

Teratogenic and antiteratogenic effects of nicotinamide derivatives in chick embryos.

Teratogenic and antiteratogenic effects of nine nicotinamide analogs in chick embryos were investigated. Further, the teratisms of 6-aminonicotinamide (6-AN), nicotinamide analogs, and an organophosphate (diazinon) were compared. White leghorn chick embryos were used. Agents were injected into the yolk of eggs on d 3 of incubation. Morphological observations were made on d 17 of incubation. Chemical names for compounds I to IX are: I, 6-dimethylaminonicotinamide; II, 6-diethylaminonicotinamide; III, 6-methylamino-3-(N-methyl)-nicotinamide; IV, 6-dimethylamino-3-pyrimidine carboximide; V, 6-(dimethylamino)-nicotinic acid; VI, 6-chloro-3-[N-(5-diethylamino)-2-pentyl]-nicotinamide; VII, 6-mercaptonicotinamide; VIII, [N-acetyl-N'-(3-pyridyl)-carbonyl]-hydrazine; IX, nicotinamide 1-N-oxide. The LD50 values in mumol per egg were as follows: 6-AN, 0.073; compound II, 0.23; compound III, 1.11; compound I, 1.32; compound VI, about 3. Compounds IV, V, VII, VIII, and IX showed no toxicity or lethality at the highest doses tested (10 mumol/egg). Among the nine nicotinamide analogs, compounds I, II, and III, which have an amino group at the 6-position of the pyridine ring, were teratogenic. Their teratogenic signs were similar to those caused by 6-AN: they showed growth retardation, anteriorly directed short legs, and coarse, dense feathering. The teratogenic effects of compounds I, II, and III were prevented by pretreatment with nicotinamide, as were the effects of 6-AN and diazinon. Among the nine analogs, only compound VIII had an antiteratogenic effect against the diazinon-induced micromelia (in which the cardinal signs were tibiotarsal angulations and poor feathering). For teratisms produced by 6-aminonicotinamide analogs and organophosphates, nicotinamide was an effective antiteratogenic agent. However, some differences in the malformations induced by both types of agents were found. We suggest that the addition of a 3-acetylpyridine type to the nicotinamide-related teratisms (6-AN type, 3-acetylpyridine type, and organophosphate type) will provide a clearer distinction among the types.

Sister-chromatid exchange and chromosomal aberrations by DHAQ and related anthraquinone derivatives in Chinese hamster ovary cells.

The induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations in Chinese hamster ovary cells by doxorubicin (adriamycin), DHAQ and several related anthraquinone derivatives was investigated. Doxorubicin, DHAQ and some of its analogues exhibited genotoxic effects at 1 nM concentration. Among them, DHAQ possessed the strongest activity. The DNA-damaging action of these substituted anthraquinone compounds correlated well with their antiproliferative effect on cells, and this action was detectable at concentrations significantly below that which caused inhibition of cell proliferation. Our data suggest that the genotoxic effects of the compounds occur prior to manifestation of their antiproliferative activity.

Induction of sister chromatid exchanges in Chinese hamster ovary cells by organophosphate insecticides and their oxygen analogs.

Induction of sister chromatid exchanges (SCEs) in cultures of Chinese hamster ovary cells by 10 anticholinesterase organophosphate insecticides was investigated. The insecticides were two phosphates (dichlorvos and dicrotophos), four sulfur-containing organophosphates (malathion, parathion, leptophos, and diazinon), and four oxygen analogs of the latter (malaoxon, paraoxon, leptophosoxon, and diazoxon). All of the compounds except diazinon induced statistically significant increases in SCE frequencies at concentrations between 0.03 and 1.0 mM. These results suggest that SCE induction is a common property of organophosphate insecticides. Compared to the sulfur-containing organophosphates, the oxygen analogs consistently produced higher SCE frequencies and had stronger antiproliferative activity. Compared to two known genotoxicants, doxorubicin and ethyl methanesulfonate, the ability of organophosphates to produce SCEs is much weaker.

Antiproliferative activity of doxorubicin and aminoanthraquinone derivatives on Chinese hamster ovary cells.

A study of the antiproliferative activity of doxorubicin and several substituted aminoanthraquinone derivatives on Chinese hamster ovary cells was conducted. Doxorubicin and a derivative each inhibited cell proliferation at low concentrations, the latter being more potent than doxorubicin. A structure-activity relationship of these compounds is discussed in connection with an earlier postulated N-O-O triangulation hypothesis.