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Biomedical subjects

H Shimada

Publications and source records attributed to H Shimada.

At least 343 records · Page 19Linked to original sources
PubMed1997

Identification of MAPKAPK homolog (MAPKAPK-4) as a myosin II regulatory light-chain kinase in sea urchin egg extracts.

We identified and cloned a homolog of mammalian mitogen-activated protein kinase-activated protein kinase (MAPKAPK)-2 and -3 from sea urchin, Hemicentrotus pulcherrimus. The obtained cDNA clone was composed of 350 amino acid residues which contain MAPK phosphorylation sites and the bipartite nuclear localization signal sites in its C-terminal domain. The clone showed 65.4 and 66.7% amino acid residue identity to human MAPKAPK-2 and -3, respectively. Phylogenetic analysis revealed that the homolog can be classified into a distinct group of MAPKAPK and, therefore, the identified homolog was designated as MAPKAPK-4. Biochemical characterization was performed using recombinant glutathione S-transferase (GST)-MAPKAPK-4 fusion protein. The protein kinase activity of GST-MAPKAPK-4 was activated by MAPK and this enabled the kinase to phosphorylate both glycogen synthase N-terminal peptide and the regulatory light chain of myosin II in vitro. Northern blot analysis showed that MAPKAPK-4 was expressed throughout the development of sea urchin embryos. These observations suggest that MAPKAPK-4 may play an important role in the regulation of myosin II activity during the development of sea urchin.

Amino Acid Sequence↗
PubMed1997

Rapid cytocidal and cytostatic chemosensitivity test by measuring total amount of mRNA.

Chemosensitivity of vinblastin, cisplatin, and mitomycin C were assessed in four different human cancer cell lines (U937, HL-60, CaR-1, and HepG2) by MTT (3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) assay and the measurement of total cytosolic poly(A) + mRNA. Results of 12 h mRNA assay with 10 x peak plasma concentration (PPC) were significantly correlated with that of standard 3 days MTT assay with 1 x PPC. Furthermore, mRNA assay was changed more significantly than MTT assay under cytostatic condition. Because of its minimum culture requirement (12 h) and broad spectrum (cytocidal and cytostatic), mRNA assay will become a useful tool for chemosensitivity test.

Drug Screening Assays, Antitumor↗
PubMed1997

Determination of ticarcillin epimers in plasma and urine with high-performance liquid chromatography.

A high-performance liquid chromatogaphic method was developed for determining the concentrations of ticarcillin (TIPC) epimers in human plasma and urine. Samples were prepared for HPLC analysis with a solid-phase extraction method and the concentrations of TIPC epimers were determined using reversed-phase HPLC. The mobile phase was a mixture of 0.005 M phosphate buffer (pH 7.0) and methanol (12:1, v/v) with a flow-rate of 1.0 ml/min. TIPC epimers were detected at 254 nm. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 1 microg/ml with a S/N ratio of 3. No peaks interfering with either of the TIPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The recovery was more than 80% for both plasma and urine samples. C.V. values for intra- and inter-day variabilities were 0.9-2.1 and 1.1-6.4%, respectively, at concentrations ranging between 5 and 200 microg/ml. The present method was used to determine the concentrations of TIPC epimers in plasma and urine following intravenous injection of TIPC to a human volunteer. It was found that both epimers were actively secreted into urine and that the secretion of TIPC was not stereoselective. Plasma protein binding was also measured, which revealed stereoselective binding of TIPC in human plasma.

Adult↗
PubMed1997

An improved method for the mouse liver micronucleus test.

We have developed a very practical method for performing the liver micronucleus test in mice. Using this method, we evaluated 11 different types of mutagens, including, 2-acetylaminofluorene, amsacrine, benzene, cyclophosphamide, diethylnitrosamine, 4-dimethylamino-3'-methylazobenzene, N-ethyl-N-nitrosourea, fluorouracil, mitomycin C, potassium chromate (VI) and selenious acid. In order to assess the sensitivity of our method, the peripheral blood reticulocyte micronucleus test was performed in the same mouse. Animals were given test chemicals once and underwent partial hepatectomy (PH) 24 h later in order to induce mitotic stimulation. Peripheral blood was sampled 0, 24, 48 and 72 h after treatment. The incidence of micronucleated hepatocytes was determined 5 days after PH. As a result, diethylnitrosamine and 4-dimethylamino-3'-methylazobenzene, known as liver carcinogens, increased the incidence of micronucleated cells in the liver only. Positive reactions for benzene, on the other hand, were found in the peripheral blood reticulocytes only. The other chemicals showed positive reaction in the liver and peripheral blood reticulocytes with almost the same maximum response of micronucleus induction. Our method was found to have the advantage over Cliets' liver micronucleus test in that it required much less time and was easier to perform procedures and highly sensitive in detecting clastogens. It can be used in combination with the peripheral blood reticulocyte micronucleus test to evaluate test chemicals in two tissues, the liver and the bone marrow, in the same animal. We propose a method of combining this test with the peripheral blood reticulocyte micronucleus test for efficient screening for the clastogenic potential of new chemicals in vivo.

Animals↗
PubMed1997

Clastogenicity and mutagenicity of hexavalent chromium in lacZ transgenic mice.

The clastogenic and mutagenic effects of the hexavalent chromium compound K2CrO4 in lacZ transgenic mice (Muta Mouse) were investigated. Male Muta mice were administered an intraperitoneal dose of 40 mg/kg of K2CrO4 once on each of 2 consecutive days. The K2CrO4 induced a significant increase in the peripheral blood micronucleated reticulocyte count. Also, K2CrO4 induced a statistically significant increase in mutant frequency in the liver but not in the bone marrow on day 7 after the second treatment. The reason for the failure to increase the mutant frequency in the bone marrow may have been the rapid cell turnover rate there. The mutation induced by K2CrO4 in the bone marrow may have occurred in more differentiated cells than stem cells, and the rapid proliferative activity may have caused a rapid decrease in mutated cells by day 7. Further study with a sampling point earlier than day 7 is needed. The results obtained in the present study indicate that K2CrO4 has clastogenic and mutagenic potential in vivo.

Animals↗
PubMed1997

Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression.

We report the establishment of stable, high-level green fluorescent protein (GFP)-expressing cell lines in vitro that permit the detection and visualization of distant micrometastases when they are implanted orthotopically in nude mice. Chinese hamster ovary cells were transfected with the dicistronic expression vector containing the humanized GFP cDNA. A stable GFP-expressing clone was selected in 1.5 microM methotrexate in vitro and injected s.c. in nude mice. Stable high-level expression of GFP was maintained in the s.c. growing tumors. To use GFP expression for metastasis studies, fragments of s.c. growing tumor, which are comprised of GFP-expressing cells, were implanted by surgical orthotopic implantation in the ovary of nude mice. Subsequent micrometastases were detected in systemic organs and could be visualized by GFP fluorescence in the lung, liver, and other organs down to the single-cell level. With this fluorescent tool, we detected and visualized for the first time tumor cells at the microscopic level in fresh viable tissue in their normal host organ. Confocal microscopy further enabled us to study physiologically relevant patterns of invasion and micrometastasis.

Animals↗
PubMed1997

The role of lymph node dissection in the treatment of gallbladder carcinoma.

BACKGROUND: Lymph node involvement is an important prognostic factor in gallbladder carcinoma (GBC). The lymph node involvement pattern, extent, and indications for systematic lymph node dissection for patients with advanced GBC were investigated. METHODS: Forty-one patients with GBC who underwent radical resection with systematic regional lymph node dissection over the past 11 years were analyzed. RESULTS: The lymph node metastasis rate was 63.4% overall, 0% in pT1 disease, 61.9% in pT2 disease, and 81.3% in pT3/pT4 disease. When reviewed according to site, the rate was 41.5% in pericholedochal lymph nodes, 22.0% in the lymph nodes around the common hepatic artery and the portal vein, 36.6% in the posterior pancreaticoduodenal lymph nodes, 28% (5/18) in the celiac lymph nodes, 19% (3/ 16) in the superior mesenteric artery (SMA) lymph nodes, and 26% (7/27) in the aortocaval paraaortic lymph nodes. Patients with severe hepatoduodenal ligament invasion had high rates of paraaortic lymph node involvement. The mortality rate was 2.4% (1 of 41 patients) and the 5-year survival rate was 33.1% overall, 100% in patients with pT1 disease, 49.8% in patients with pT2 disease, and 0% in patients with pT3/pT4 disease. The 5-year survival rate for pT2 disease according to lymph node involvement was 72.7% in patients with pN0+ pN1+ positive posterior pancreaticoduodenal lymph nodes and positive lymph nodes around the common hepatic artery in the N2 patients and 0% in the patients with positive celiac and SMA lymph nodes in the N2 patient group or the positive paraaortic lymph node group (P < 0.05). CONCLUSIONS: These results suggest that systemic dissection of N1 lymph nodes, posterior pancreaticoduodenal lymph nodes, and lymph nodes around the common hepatic artery and the portal vein in N2 patients is necessary to improve the prognosis of those patients with pT2 disease without moderate or severe hepatoduodenal ligament invasion.

Adult↗
PubMed1997

Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (groups 1, 2A and 2B) the summary report of the 6th collaborative study by CSGMT/JEMS MMS. Collaborative Study of the Micronucleus Group Test. Mammalian Mutagenicity Study Group.

To assess the correlation between micronucleus induction and human carcinogenicity, the rodent micronucleus assay was performed on known and potential human carcinogens in the 6th MMS/CSGMT collaborative study. Approximately 100 commercially available chemicals and chemical groups on which there was little or no micronucleus assay data were selected from IARC (International Agency for Research on Cancer) Groups 1 (human carcinogen), 2A (probable human carcinogen) and 2B (possible human carcinogen). As minimum requirements for the collaborative study, 5 male mice were treated by intraperitoneal injection or oral gavage once or twice with each chemical at three dose levels, and bone marrow and/or peripheral blood was analyzed. Five positives and 2 inconclusives out of 13 Group 1 chemicals, 7 positives and 5 inconclusives of 23 Group 2A chemicals, and 26 positives and 6 inconclusives of 67 Group 2B chemicals were found. Such low positive rates were not surprising because of a test chemical selection bias, and we excluded well-known micronucleus inducers. The overall evaluation of the rodent micronucleus assay was based on the present data combined with published data on the IARC carcinogens. After merging, the positive rates for Groups 1, 2A and 2B were 68.6, 54.5 and 45.6%, respectively. Structure-activity relationship analysis suggested that the micronucleus assay is more sensitive to the genetic toxicity of some classes of chemicals. Those to which it is sensitive consist of (1) aziridines and bis(2-chloroethyl) compounds; (2) alkyl sulfonate and sulfates; (3) acyl-type N-nitroso compounds; (4) hydrazines; (5) aminobiphenyl and benzidine derivatives; and (6) azo compounds. Those to which it is less sensitive consist of (1) dialkyl type N-nitroso compounds; (2) silica and metals and their compounds; (3) aromatic amines without other functional groups; (4) halogenated compounds; and (5) steroids and other hormones. After incorporation of structure-activity relationship information, the positive rates of the rodent micronucleus assay became 90.5, 65.2 and 60.0% for IARC Groups 1, 2A and 2B, respectively. Noteworthy was the tendency of the test to be more sensitive to those carcinogens with stronger evidence human carcinogenicity.

Animals↗
PubMed1997

Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse).

The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse). Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection. Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis. Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue. Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging. The resultant phages were allowed to infect to E. coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside. Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times. ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF. MMS was not mutagenic in either tissue. The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice. These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis. On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system.

Animals↗
PubMed1997

Germ cell mutagenesis in lacZ transgenic mice treated with methyl methanesulfonate.

Mutagenesis induced by methyl methanesulfonate (MMS), a germ cell mutagen, in the testis and the sperm isolated from epididymis and vas deferens have been investigated using lacZ transgenic mice (Muta Mouse). Male Muta Mice were injected intraperitoneally with MMS at a dose of 80 mg/kg, a potent dominant lethal dose. Animals were killed on days 3 and 7 (Experiment 1) or days 10 and 14 (Experiment 2) after the treatment. Mutant frequencies (MFs) in the testis, sperm and spleen (Experiment 2 only) were analyzed by the positive selection system using E. coli C (GalE-) strain and phenyl beta-D-galactoside. The spontaneous MFs in the testis and sperm were 2.0-3.1 x 10(-5). No induction of mutation in the testis or sperm of the MMS-treated groups was observed at any sampling point. In the spleen, the spontaneous MF was approximately twice as high as that in the germ cells although the MF at each sampling point was almost the same as the spontaneous MF. MMS is known as a potent clastogen from the results of the dominant lethal assay and the micronucleus assay. The reason for the discrepancy between the results of these assays and the present results may have been insensitivity of the in vitro packaging to large deletion due to the failure to rescue the large deleted gene. It is suggested that the transgenic mouse assay using the in vitro packaging can not replace the dominant lethal assay in the case of MMS.

Animals↗
PubMed1997

Two isoforms of orthodenticle-related proteins (HpOtx) bind to the enhancer element of sea urchin arylsulfatase gene.

The sea urchin (Hemicentrotus pulcherrimus) arylsulfatase (HpArs) gene, expressed specifically in aboral ectoderm, contains a 229-bp enhancer in its first intron that is required for the activation of HpArs gene expression. Deletion analysis shows that a tandem repeat of orthodenticle-related protein (Otx) binding sites are responsible for the activity of this enhancer. Gel mobility shift analysis reveals that three types of Otx-proteins, which show different mobilities in gel shift assays, form complexes with the enhancer. Band I appears before hatching and gradually decreases by the gastrula stage. Band III appears at the blastula stage and Band II appears at the mesenchyme blastula stage; the levels of Band II and III remain constant until the gastrula stage. Two distinct types of HpOtx cDNA clones have been isolated from cDNA libraries of unfertilized eggs and gastrulae. Nucleotide sequences of the homeobox and downstream regions are well conserved in the two types of HpOtx cDNAs, while the region upstream from the homeobox has different nucleotide sequences. By genomic Southern blot analysis, only a single copy of HpOtx gene is detectable in the Hp genome, making it likely that two HpOtx isoforms are generated from the same gene. Results from Northern blot analysis confirm the presence of two types of HpOtx transcripts. Transcriptional regulation of the HpArs gene may, in part, be carried out through switching of Otx isoforms.

Amino Acid Sequence↗
PubMed1997

Effects of dithiocarbamates on toxicity of cadmium in rat primary hepatocyte cultures.

The protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD) on the toxicity of Cd in the rat primary hepatocyte cultures were studied. Cytotoxicity was assessed by measuring cell viability, extra cellular lactic dehydrogenase (LDH) activity, and intracellular lipid peroxidation and active oxygen species. Primary hepatocyte cultures were treated with 109CdCl2 (5, 10 or 50 microM Cd and 1.7 KBq of 109Cd/well) for 30 min or 4 h. BGD or HBGD was added to the culture medium to make the final concentration of 100 microM and incubated for 4.5 h in 30 min Cd exposure or 1 h in 4 h Cd exposure. Decreases in the hepatocyte viability caused by all Cd exposure concentrations were significantly prevented by treatment with BGD or HBGD. The treatment with the chelating agents for 4.5 h after Cd exposure for 30 min significantly prevented increases in extracellular LDH activity. Increases in the lipid peroxidation in hepatocytes exposed to Cd for 30 min or 4 h were prevented significantly by treatment with BGD or HBGD for 4.5 h or 1 h, respectively. Moreover, the increases in the level of active oxygen species caused by Cd exposure for 30 min were significantly prevented by treatment with the chelating agents for 1.5 h. These findings suggest that BGD and HBGD protect against the cytotoxicity of Cd in rat primary hepatocyte cultures and that the protective effects of chelating agents presumably result from a decrease in the Cd level, the effective sequestration of the reactive Cd ion, and the direct preventive effect on the active oxygen species in the hepatocytes.

Animals↗
PubMed1997

Testosterone pretreatment mitigates cadmium toxicity in male C57 mice but not in C3H mice.

Previous work has indicated that testosterone pretreatment protects against cadmium-induced toxicity in male rats while other data indicate that pretreatment of mice with testosterone offers no such protection against cadmium. Since cadmium toxicity may vary widely with species and strain, we examined the effect of testosterone pretreatment on cadmium toxicity in two strains of mice, one that is sensitive (C3H) and one that is resistant (C57) to cadmium toxicity. A single sc injection of 20 micromol CdCl2/kg to C3H mice or 45 micromol CdCl2/kg to C57 mice proved very toxic, causing 50%, and 44% mortalities, respectively. However, when C57 mice were pretreated with testosterone (5 mg/kg, s.c., at - 48, - 24, and 0 h) prior to cadmium (45 micromol/kg), complete resistance to cadmium-induced lethality developed. Testosterone had no effect on cadmium-induced lethality in C3H mice. Testosterone prevented extensive hepatocellular damage caused by cadmium in C57 mice and also significantly reduced cadmium-induced elevations in serum lactate dehydrogenase (LDH) activity and blood urea nitrogen (BUN), which are indicators of hepatic and renal function, respectively. The toxicokinetics of cadmium were apparently not affected by testosterone pretreatment, as the distribution of cadmium to liver in either strain was unchanged by the steroid. Cadmium-induced metallothionein (MT) levels in liver and kidney of C57 mice were increased in testosterone-pretreated mice given the higher doses of metal but no such enhancement of MT synthesis occurred in C3H mice. This increase in MT may provide some level of protection against cadmium toxicity in the C57 mice. These results indicate that testosterone pretreatment prevents toxicity of cadmium in male C57 mice, possibly through enhancement of MT synthesis, but has no effect in male C3H mice.

Animals↗
PubMed1997

Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts.

The crt gene clusters responsible for the biosynthesis of carotenoids such as lycopene, beta-carotene and astaxanthin have been isolated from carotenogenic bacteria such as Erwinia species and the marine bacterium Agrobacterium aurantiacum. The functions of the individual genes have been identified. The first substrate of the enzymes encoded by the Erwinia crt clusters is farnesyl pyrophosphate which is not only the precursor for carotenoid biosynthesis but also sterols, dolichols and other numerous isoprenoid compounds. Escherichia coli does not naturally synthesize carotenoids, but by using the carotenogenic genes recombinant strains accumulating lycopene, beta-carotene and astaxanthin have been produced. Other non-carotenogenic bacteria such as Zymomonas mobilis have also been engineered to produce beta-carotene by the introduction of the corresponding crt genes. A gene capable of enhancing carotenoid levels in E. coli has also been isolated from cDNA libraries of the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. This gene has been found to encode an isopentenyl pyrophophate isomerase. It has further been shown that the edible yeasts Candida utilis as well as Saccharomyces cerevisiae, which possess no carotenoid biosynthetic pathway, acquire the ability to produce carotenoids, when the carotenogenic genes are expressed under the control of yeast-derived promoters and terminators. It has been observed in the yeasts S. cerevisiae and C. utilis carrying the lycopene biosynthesis genes that ergosterol content is decreased by 10 and 35%, respectively. It is therefore likely that the carbon flux for the ergosterol biosynthesis has been partially directed from farnesyl pyrophosphate to a new pathway for the lycopene biosynthesis. Further, the expression of a truncated gene which codes for the catalytic domain of the endogenous 3-hydroxy-3-methylglutaryl coenzyme. A reductase, has been found to be effective for enhancing carotenoid levels in the yeast C. utilis.

Carotenoids↗
PubMed1997

Primary and metastatic rhabdomyosarcoma in the breast: neoplasms of adolescent females, a report from the Intergroup Rhabdomyosarcoma Study.

UNLABELLED: The occurrence of rhabdomyosarcoma (RMS) primary in or metastatic to breast has been regarded as an uncommon event, associated with an unfavorable outcome. Records of 26 patients with diagnoses of breast RMS, either primary or secondary, entered in the Intergroup Rhabdomyosarcoma Study (IRS) (1972-1992) were reviewed and compared with data regarding 47 similar patients in published reports. Of the 26 IRS cases, the histologic subtype was alveolar in 24, embryonal in 1, and not determined in 1. All were female with ages ranging from 11.5 to 20.2 years (median, 15.2 years; mode, 14-16 years). This compact age distribution of both primary (n = 7) and metastatic (n = 19) breast RMS was seen in previously reported series. Among the 19 cases of RMS with initial dissemination to breast, primary tumor sites, were extremity (n = 8), nasopharynx/paranasal sinuses (n = 7), and trunk (n = 4). IRS treatment was risk-based according to site and extent of disease. Four of 7 patients with primary RMS remain disease free 2.9 to 7 years post diagnosis. Among 19 patients with RMS initially metastatic to breast, including 7 in IRS clinical group IV at original diagnosis, three are disease free at 7.6, 15.7 and 17.0 years. CONCLUSIONS: primary or metastatic RMS in breast is almost confined to adolescent females having tumors with alveolar histology. Approximately one-half of the patients with primary breast disease and 15% of those with metastatic breast disease as an initial recurrence are long-term survivors.

Adolescent↗
PubMed1997

Amyloid beta-protein deposition in the leptomeninges and cerebral cortex.

To further investigate the process of amyloid beta-protein (Abeta) deposition, we determined, using sensitive enzyme immunoassays, the levels of Abeta40 and Abeta42 (Abetas) in the soluble and insoluble fractions of the leptomeninges (containing arachnoid mater and leptomeningeal vessels) and cerebral cortices from elderly control subjects showing various stages of Abeta deposition and from patients affected by Alzheimer's disease (AD). In both locations, insoluble Abeta levels were higher by orders of magnitude than soluble Abeta levels. Soluble Abeta levels in cortices were much lower than those in leptomeninges. In insoluble Abeta in the cortex, Abeta42 was by far the predominant species, and Abeta42 in AD cortices was characterized by the highest degree of modifications in the amino terminus. In contrast, this Abeta42 predominance was not observed in insoluble Abeta in the leptomeninges, which were found to be able to accumulate Abetas to an extent similar to that in the cortex, on a weight basis. The levels of insoluble Abeta in the leptomeninges or cortex generally correlated with the degree of cerebral amyloid angiopathy or the abundance of senile plaque, respectively. However, the presence of plaque-free cortical samples showing significant levels of insoluble Abeta42 suggests that biochemically detectable Abeta accumulation precedes immunocytochemically detectable Abeta deposition in the cortex.

Aged↗
PubMed1997

Progesterone pretreatment enhances cellular sensitivity to cadmium despite a marked activation of the metallothionein gene.

Previously, we found that in vivo pretreatment with progesterone markedly increased cadmium lethality in rats, apparently by enhancing cadmium-induced hepatonecrosis. Therefore, the present study was designed to investigate this phenomenon at the molecular level in an in vitro system. TRL-1215 rat liver cells were exposed to various concentrations of progesterone (0, 1, 10, and 100 microM) for 24 hr and subsequently exposed to cadmium (0, 1, 5, 10, and 50 microM; as CdCl2) for an additional 24 hr. Although the levels of progesterone used were essentially nontoxic, progesterone pretreatment resulted in a concentration-dependent increase in sensitivity to cadmium as assessed by loss of mitochondrial enzyme activity (tetrazolium-based dye assay) and loss of cytosolic enzyme activity (glutamic oxaloacetic transaminase). The effects of progesterone treatment on intracellular levels of metallothionein (MT), an inducible metal-binding protein generally associated with cadmium tolerance, were also measured. Progesterone (100 microM) alone increased MT levels 2.4-fold, while cadmium (10 microM) alone resulted in a 7-fold increase over control. Progesterone pretreatment followed by cadmium exposure caused a marked, 16-fold induction in MT synthesis, a level of activity that has been associated with acquired tolerance to cadmium. In addition, progesterone pretreatment clearly induced transcription of the MT gene as evidenced by enhanced cadmium-induced accumulation of cellular MT mRNA. Progesterone pretreatment had no effect on the level of glutathione, a cellular thiol thought to be important in detoxication of cadmium prior to MT gene activation and MT protein accumulation, or on cellular accumulation of cadmium during the initial 3 hr of exposure to the metal. The proportion of total cellular cadmium bound to MT in cells pretreated with progesterone was greater than that in the cells treated with cadmium alone, indicating an enhanced sequestration of the metal by MT after pretreatment. These results indicate that progesterone, at nontoxic levels, markedly exacerbates cadmium toxicity at the cellular level in liver cells. This is in accord with the observed progesterone-induced enhancement of the hepatotoxic effects of cadmium in vivo. The observed facilitation of cytotoxicity is not based in altered toxicokinetics of cadmium and occurs despite a pronounced activation of the MT gene resulting in an enhanced sequestration of cadmium by MT. The mechanism by which progesterone enhances cadmium toxicity deserves further study.

Animals↗