Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay.

Evaluation of the mutagenic potential of ubidecarenone using three short-term assays.

Addition of ubidecarenone, coenzyme Q(10) (CoQ(10)), to foods has been proposed for its nutritive value. Ubidecarenone is present naturally in a number of foods, including meats (e.g., beef, chicken) and fish (e.g., herring, rainbow trout), and on average, people are estimated to consume 2-20 mg/day of this metabolically important substance. Currently, relatively little formal evidence regarding the safety of ubidecarenone has been identified in the toxicology literature, despite its consumption by humans for centuries without reported notable adverse effects. As such, a series of toxicological studies, including mouse bone marrow micronucleus, chromosomal aberration, and bacterial reverse mutation tests, were conducted to evaluate the in vivo and in vitro mutagenic potential of CoQ(10). The test article, ubidecarenone, was devoid of clastogenic activity when administered orally to mice at doses up to 2000 mg/kg/day. In addition, the test article did not induce chromosomal aberration in CHL/IU cells exposed to concentrations as great as 5.0 mg/ml, nor did it induce reverse mutations in Salmonella typhimurium and Escherichia coli at concentrations as great as 5000 microg/plate.

Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice.

Accumulating evidence indicates that gap junctions play an important role in the maintenance of normal cell growth, so that genes for the connexin gap junction proteins form a family of tumor-suppressor genes. Although mice from which nine types of connexin gene are deleted have been established, little information from carcinogenesis experiments with these mice is available. We have previously found several mutant forms of connexin 32 (Cx32) to be able to inhibit, in a dominant-negative manner, gap junctional intercellular communication (GJIC) exerted by wild-type Cx32. By introducing a gene for such a dominant-negative Cx32 mutant expressed under the control of a liver-specific albumin gene promoter, we have generated transgenic mouse lines in which the function of Cx32 is down-regulated only in the liver. Although GJIC was diminished in the transgenic liver as expected, the reduced GJIC did not affect viability nor the number of spontaneous liver tumors. Although susceptibility to diethylnitrosamine-induced hepatocarcinogenesis was significantly elevated in the transgenic mice, liver regeneration after partial hepatectomy was delayed compared with wild-type mice, suggesting that gap junctions function not only to suppress excessive cell growth but also to promote cell proliferation when necessary for normal function of tissues. Although the phenotype of Cx32-deficient mice was similar to that of the transgenic mice, the former showed more drastically altered phenotypes, i.e. increased BrdU incorporation in the quiescent liver and development of spontaneous liver tumors. We also established 3T3 fibroblasts from embryos lacking the Cx43 gene and characterized their growth. These fibroblasts showed no difference from the wild type in growth characteristics. From these and other studies, we suggest that gap junctions do not necessarily suppress cell growth but support an optimal growth rate.

Induction of skin papillomas, carcinomas, and sarcomas in mice in which the connexin 43 gene is heterologously deleted.

It has been suggested that blocked gap junctional intercellular communication plays a crucial part in multistage carcinogenesis. The mouse skin tumor-promoting phorbol esters are potent inhibitors of gap junctional intercellular communication and this inhibition is considered to be a mechanism by which clonal expansion of "initiated" cells is promoted. We examined whether mice in which the gene for a gap junction protein, connexin 43, is heterozygously deleted are more susceptible to chemical carcinogenesis; connexin 43 is expressed in the basal cell layer and the dermis of the skin. When the back skin was painted with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate, the incidence and yields of both papillomas and carcinomas were similar in connexin 43+/- and connexin 43+/+ mice; for this experiment, the original mice with C57BL/6 genetic background was crossed with CD1 strain for three generations. Subcutaneous injection of 7, 12-dimethylbenz[a]anthracene resulted in induction of fibrosarcomas in connexin 43+/- and connexin 43+/+ mice to a similar extent. All papillomas and carcinomas induced with 7, 12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate contained the 7,12-dimethylbenz[a] anthracene-specific mutation in the ras gene (A to T transversion at the 61st codon). About 50% of fibrosarcomas also contained this mutation, but in the Ki-ras gene; there was no difference in the prevalence of this mutation in tumors from connexin 43+/- and connexin 43+/+ mice. None of the tumors examined, however, showed any mutation in the connexin 43 gene. These results suggest that the deletion of one allele of the connexin 43 gene does not significantly contribute to, nor alter, the molecular events involved in skin carcinogenesis. These results are compatible with previous observations that nongenetic disruption of function rather than mutations of connexins, commonly occurs in cancer cells.

Connexins in tumour suppression and cancer therapy.

Malignant cells usually show altered gap junctional intercellular communication and are often associated with aberrant expression or localization of connexins. Transfection of connexin genes into tumorigenic cells restores normal cell growth, suggesting that connexins form a family of tumour suppressor genes. Some studies have also shown that specific connexins may be necessary to control growth of specific cell types. Although we have found that genes encoding connexin32 (Cx32; beta 1), Cx37 (alpha 4) and Cx43 (alpha 1) are rarely mutated in tumours, our recent studies suggest that methylation of the connexin gene promoter may be a mechanism by which connexin gene expression is down-regulated in certain tumors. We have produced various dominant negative mutants of the genes encoding Cx26 (beta 2), Cx32 and Cx43, some of which prevent the growth control exerted by the corresponding wild-type genes. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through gap junctions. Recently, we and others have shown that gap junctional intercellular communication is responsible for the bystander effect seen in herpes simplex virus thymidine kinase/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.

Growth control of 3T3 fibroblast cell lines established from connexin 43-deficient mice.

Connexins are considered to be involved in cell growth control, on the basis of studies mainly with tumorigenic cells. To study the role of connexin genes in normal cell growth control, we established fibroblast cell lines from connexin 43 (Cx43)-deficient mice and characterized their growth. Embryonic fibroblasts from wild-type mice (Cx43+/+) and those with heterozygous (Cx43+/-) and homozygous (Cx43+/-) deficiencies of the Cx43 gene were cultured and passaged by a 3T3 protocol (every 3 d, 3 x 10(5) cells/60-mm dish). All cell lines showed a growth crisis during passages 6-15 and then started to grow well. All cell lines grew at similar rates under the 3T3 protocol, but Cx43-deficient (Cx43-/-) cell lines tended to grow faster when they were plated at 10(5) cells per dish. Cx43-/- cells did not express Cx43 and showed little gap-junctional intercellular communication (GJIC), confirming that Cx43 is the major connexin responsible for GJIC of these fibroblasts. While all Cx43+/+ and Cx43+/- cell lines expressed Cx43 protein, some of them showed very little GJIC. Those cell lines with high GJIC showed higher levels of the P2 form of Cx43 protein, and more Cx43 was localized in the plasma membrane than in cell lines with lower GJIC levels. We investigated effects of serum concentration on cell growth in these cell lines. Although different cell lines responded differentially to these agents, there was no clear relationship between Cx43 expression and cell growth stimulation by them. This suggests that Cx43 expression alone is not a strong regulator of mouse fibroblast growth.

Comparative studies of MCL-5 cells and human lymphocytes for detecting indirect-acting clastogens.

The MCL-5 cell line was established from human lymphoblastoid TK+/- cells transfected with cDNAs of human cytochrome P450s (CYP1A2, CYP2A6, CYP2E1, and CYP3A4) and microsomal epoxide hydrolase. The TK+/- cells constitutively express a relatively high level of endogenous CYP1A1. To study metabolic activities to indirect-acting clastogens, MCL-5 cells were treated with four clastogens, i.e. aflatoxin B1 (AFB1), diethylnitrosamine (DEN), cyclophosphamide (CPA), and 7,12-dimethylbenz[a]anthracene (DMBA). Human lymphocytes from peripheral blood were used as control cells under the assay conditions with or without induced rat liver metabolic activation (S9). All chemicals tested without S9 induced chromosomal aberrations (CA) in MCL-5 cells but not in human lymphocytes. All chemicals induced CA in both cell types in the presence of S9.

Re-evaluation of chromosomal aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens.

In a collaborative study organized under the JEMS MMS, nine mouse lymphoma assay (MLA) "unique positive' NTP rodent carcinogens were re-evaluated by an in vitro chromosomal aberration assay using Chinese hamster lung fibroblast cells (CHL/IU). Six of nine chemicals induced chromosomal aberrations; bromodichloromethane, chlorendic acid and isophorone induced structural aberrations, and chlorodibromomethane, pentachloroethane and 1,1,1,2-tetrachloroethane induced numerical aberrations (polyploidy). These six chemicals, therefore, are not uniquely positive in the MLA. The difference between the NTP results and ours might be due to the use of different cell lines and protocols, and in some cases, to different interpretations of polyploidy. The remaining three chemicals, benzyl acetate, cinnamyl anthranilate and trichloroethylene, were negative in this study.

Detection of neocarzinostatin-induced translocations in human sperm chromosomes using fluorescence in situ hybridization of chromosome 2.

Mature sperm and late spermatid are known to be sensitive stages to clastogens in mammalian spermatogenesis. Certain types of chromosomal damage induced in these stages will pass to successive generations as heritable translocations. In the present study, we employed whole chromosome 2 painting with the fluorescence in situ hybridization (FISH) technique to detect the chemically induced translocations in human sperm. Mature human sperm were treated in vitro with an antitumor drug, neocarzinostatin (NCS), and fertilized in vitro with golden hamster oocytes. Sperm pronuclear chromosome slides were prepared at the first cleavage metaphase. To compare the characteristics of translocations between somatic and germ cells, human lymphocytes in peripheral blood treated with NCS in vitro were analyzed at first round metaphase after PHA-stimulation. From the analysis of translocations by whole chromosome 2 painting, frequencies of the haploid genomic translocations (FhG) were predicted for both sperm and lymphocytes. At 1.0 micrograms/ml, the actual percentages of sperm and lymphocytes with chromosome 2 translocations were almost identical (11.9% and 12.0%). At the same dose, however, the FhG of the sperm (1.15) was considerably higher than that of the lymphocytes (0.58), indicating that complex translocations having two or more rearranged sites were induced by NCS more frequently in sperm than in lymphocytes.

Germanium poisoning: clinical symptoms and renal damage caused by long-term intake of germanium.

We report five patients who have taken inorganic germanium preparations over a prolonged period. In all cases, the renal function deteriorated with no proteinuria or hematuria. Histological examination of the kidneys showed widespread tubular degeneration and interstitial fibrosis with minor glomerular abnormalities. Most patients had gastrointestinal symptoms such as vomiting, anorexia and weight loss; one patient had peripheral neuropathy and myopathy. A considerable amount of germanium was detected in the hair or nails of these patients. These cases clearly show that abuse of inorganic germanium compounds can induce renal damage with various extrarenal manifestations.

Progression of experimental focal glomerulosclerosis in the spontaneously hypertensive rat.

To study the influence of hypertension on the progression of focal glomerulosclerosis (FGS), we produced an experimental model of FGS in spontaneously hypertensive rats (SHRs) by the combined administration of puromycinaminonucleoside (AMNS) and protamine sulfate (PS). SHRs and normotensive Wistar Kyoto rats as a control strain were given daily injections of subcutaneous AMNS (1 mg/100 gm body weight) and intravenous PS (two separated doses of 2.5 mg/100 mg body weight) for 4 days; they were killed on day 80 after three series of injections at 10-day intervals. The levels of urinary protein, serum creatinine, and urea nitrogen in SHRs given AMNS and PS were elevated throughout the experiment and were significantly higher than these levels in other control groups on day 80. Histology in SHRs given AMNS and PS showed advanced FGS associated with glomerular hypertrophy and widespread interstitial fibrosis. Most small arteries and arterioles showed "onion peel" thickening and fibrinoid necrosis of the intima, which is characteristic of malignant arteriosclerosis. We observed that the gradient of glomerulosclerosis increased from superficial to deep cortical zones; this phenomenon had often been reported in human FGS. However, these distinguished lesions were not found in control groups. Therefore, it is suggested that systemic hypertension is one of the deleterious factors enhancing histologic and functional deterioration in FGS.

The effect of protamine on serum complement activity in aminonucleoside nephropathy.

Combined administration of protamine sulfate (Ps) and aminonucleoside (AN) to rats causes severe nephrotic syndrome and, histologically, focal glomerular sclerosis. These changes are more distinct than those produced by AN alone. While the exact mechanism of Ps acting on AN nephropathy is not known, marked hypocomplementemia was observed regularly after the Ps + AN injection, suggesting that complement is activated and consumed in the kidney. It is suggested that complement may play an important role in AN + Ps nephropathy.

The effect of protamine on proteinuria and glomerular sclerosis in aminonucleoside nephropathy.

An administration of protamine sulfate combined with aminonucleoside of puromycin (AN) could produce in rats severe nephrotic syndrome and histological changes similar to focal glomerular sclerosis more distinctly than AN alone. This may be attributed to the action of protamine which seemed to enhance the effect of AN, causing polyanion loss at the glomerular basement membrane.