Chemical induction of presumed dominant-lethal mutations in postcopulation germ cells of mice. I. Relative sensitivity between pre- and postcopulation germ cells to isopropyl methanesulfonate

Females from ($\text{C}\text{3}\text{H}\text{×}\text{IOI}\text{)}\text{F}{}_1$ and a mixed stock were injected intraperitoneally with either 25 or 50 mg/kg isopropyl methanesulfonate (IMS) or Hanks' solution 4.5 h after the midpoint of the dark period during which mating occurred. It was determined that at the time of treatment the great majority of oocytes were undergoing second meiotic division. For comparison, the same doses of IMS were given to females treated within 3.5 days prior to mating (predominantly dictyate oocytes) or to males treated within 4.5 days prior to mating (sperm in vas and epididymis). The frequencies of presumed dominant lethals induced by 50 mg/kg IMS in sperm treated in vas and epididymis, dictyate oocytes, and germ cells in mated females are 22%, 19%, and 79%, respectively, for (C3H × IOI)F₁ and 26%, 30%, and 76% for the other stock. Clearly, in both stocks, effects in mated females, when both female and male germ cells were treated, are relatively much higher than the added effects on dictyate oocytes and spermatozoa. This is also true for the 25 mg/kg dose.     

Vitamin E as a novel enhancer of macroautophagy in rat hepatocytes and H4-II-E cells

Autophagy is an intracellular bulk degradation process induced by nutrient starvation, and contributes to macromolecular turnover and rejuvenation of cellular organelles. We demonstrated that vitamin E was a novel nutritional enhancer of autophagy in freshly isolated rat hepatocytes and rat hepatoma H4-II-E cells. Supplementation of fresh hepatocytes with vitamin E (up to 100 μM) increased proteolysis significantly in the presence or absence of amino acids in a dose-dependent manner. The cytosolic LC3 ratio, a newly established index of autophagic flux, was significantly increased by vitamin E, strongly suggesting that the possible site of action is the LC3 conversion step, an early step in autophagosome formation. A typical antioxidant, α-lipoic acid, exerted autophagy suppression, while H₂O₂ stimulated autophagy. It is conceivable that autophagy was stimulated by oxidative stress and this stimulation was cancelled by cellular antioxidative effects. However, in our studies, vitamin E could have enhanced autophagy over-stimulation by H₂O₂, rather than suppress it. From these results, using a new cytosolic LC3 ratio, vitamin E increases autophagy by accelerating LC3 conversion through a new signaling pathway, emerging as a novel enhancer of autophagy.     

Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore

After a brief review of the early history of mitochondrial electrophysiology, the contribution of this approach to the study of the mitochondrial permeability transition (MPT) is recapitulated. It has for example provided evidence for a dimeric nature of the MPT pore, allowed the distinction between two levels of control of its activity, and underscored the relevance of redox events for the phenomenon. Single-channel recording provides a means to finally solve the riddle of the biochemical entity underlying it by comparing the characteristics of the pore with those of channels formed by candidate molecules or complexes. The possibility that this entity may be the protein import machinery of the inner mitochondrial membrane is emphasized.     

Apparent lack of correlation between the loss of cytochrome P-450 in hepatic parenchymal cell culture and the stimulation of haem oxygenase activity

The hypothesis that the stimulation of haem oxygenase activity in cultured adult rat liver parenchymal cells is intimately associated with the accelerated breakdown of the haemoprotein cytochrome P-450 was examined. Even though the time course of the loss of cytochrome P-450 and the stimulation of haem oxygenase activity were found to be compatible with this hypothesis, further work however showed that high levels of cytochrome P-450 could be maintained in liver cell culture in the face of high haem oxygenase activities.     

Binding versus biological activity of Clostridium perfringens enterotoxin in Vero cells.

Cells resistant to $\text{Clostridium perfringens}$ enterotoxin were selected from cultures of highly sensitive Vero (African green monkey kidney) cells. Studies were done with the sensitive and resistant cells to determine the relationship between binding and biological activity. Binding studies using ¹²⁵I-enterotoxin revealed the apparent existence of high and low affinity binding sites for the enterotoxin on both cell types. The binding site density on resistant cells was found to be $\text{1}\text{10}$ that of sensitive cells. It was found that, even with high doses of enterotoxin, only partial affect upon DNA synthesis, membrane permeability, and plating efficiency was noted in resistant cells. It is concluded that without specific binding there is little or no ability of the enterotoxin to effect biological activity in cells.     

Ethylation pf DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals

The molecular dosimetry of ethyl methanesulfonate (EMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 200 mg/kg of [³H]EMS and the ethylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 2-week period. The ethylations per sperm head closely paralleled the dominant-lethal frequency curve for EMS, reaching a maximum of 5 to 6.5 million ethylations per vas sperm head at 8 to 10 days after treatment. Ethylation of sperm DNA was greatest at 4 h after treatment, with 5.7 ethylations/10⁵ deoxynucleotides, and gradually decreased to 2.2 ethylations/10⁵ deoxynucleotides at 15 days after treatment. The ethylation of sperm DNA did not increase in the germ-cell stages most sensitive to EMS, ans was not correlated with the dominant-lethal frequency curve for EMS. However, ethylation of sperm protamine did increase in the germ-cell stages most sensitive to EMS, and showed an excellent correlation with the incidence of dominant lethals produced by EMS in the germ cells.A model is presented to explain, at a molecular level, how dominant lethals may be induced in mouse germ cells by EMS. Ethylation by cysteine sulfhydryl groups contained in mouse-sperm protamine could block normal disulfidebond formation, preventing proper chromatin condensation in the sperm nucleus. Stresses in the chromatin structure could then eventually lead to chromosome breakage, with resultant dominant lethality.     

Differences in sialyl transferase activity and in concanavalin-A agglutination between T and B lymphoblastoid cell lines.

The concanavalin A agglutination patterns, sialyl transferase activity and sialic acid content were studied for cultured lymphoblastoid cell lines possessing either T or B surface markers. Concanavalin A caused marked agglutination of the two B cell lines studied, Raji and SB, while the two T cell lines, HSB-2 and CCRF-CEM, failed to agglutinate with this lectin. The surface sialyl transferase activity of the two B lines was significantly higher than on the two T lines studied. In contrast, the total cellular sialic acid content or the surface sialic acid that was released from the T and B cell lines by neuraminidase was not significantly different. This study indicates that T and B lymphoblastoid cells possess different levels of surface located sialyl transferase activity and display different agglutination patterns with Con A. Perhaps these assays can be of value in differentiating various classes of neoplastic lymphoid cells.     

Investigation of styrene in the liver perfusion/cell culture system. No indication of styrene-7,8-oxide as the principal mutagenic metabolite produced by the intact rat liver

Mutagenic effect of styrene and styrene-7,8-oxide was studied with the isolated perfused rat liver as metabolizing system and Chinese hamster V79 cells as genetic target cells. Styrene-7,8-oxide which is mutagenic per se was rapidly metabolized by the perfused rat liver. Thus no mutagenic effect was detected neither in the perfusion medium nor in the bile. However when styrene was added to the perfusion system, an increase in V79 mutants was observed regardless of where in the circulating perfusion medium the V79 cells were placed: the same effect was obtained with V79 cells close to the liver as well as at a distance from the liver. No mutagenic effect was observed in the bile. Simultaneous analysis of the styrene-7,8-oxide concentration in the perfusion medium, suggest that this metabolite is not the cause of the mutagenic effect observed during perfusion with styrene.The effect of the two test compounds on some liver functions was also studied. Both styrene and styrene-7,8-oxide changed the bile flow without affecting bile acid secretion: styrene caused a reduction in bile flow as compared to control perfusions and styrene-7,8-oxide increased the bile flow. Styrene, but not styrene-7,8-oxide, reduced gluconeogenesis from lactate. Styrene had no effect on the liver's capacity to incorporate amino acids into plasma proteins, whereas styrene-7,8-oxide reduced the amino acid incorporation. The microsomal cytochrome P-450 content was not affected by the two test compounds. No alteration in microsomal N- and C-oxygenation of N, N-dimethylaniline (DMA) was observed with styrene-7,8-oxide or the lower styrene dose used (240 μmol), whereas the higher styrene concentration (480 μmol) reduced N-oxygenation and thus also the total DMA metabolism.It is suggested that the results on styrene and styrene-7,8-oxide found here using the liver perfusion/cell culture system mimic the metabolism expected to be found in the intact animal, thus indicating that styrene-7,8-oxide is not the principal mutagenic metabolite of styrene in vivo.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.