Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

Publisher Summary This chapter presents the assay of glutathione (GSH), glutathione disulfide (GSSG), and glutathione mixed disulfides (GSSR). The sum of the reduced and oxidized forms of glutathione can be determined by using a kinetic assay in which catalytic amounts of GSH or GSSG and glutathione reductase bring about the continuous reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by nicotinamide adenine dinucleotide phosphate (NADPH). The sensitivity of the assay may be enhanced by measuring NADPH fluorometrically. There are other two enzymatic assay available. In the first assay, the formation of the reduced glutathione using glyoxalase I can be monitored directly at 240 nm, or at very low levels of GSH, a dual wavelength spectrophotometer can be used at the wavelength-pair 240–270 nm with the same extinction coefficient. The second assay involves O-phthaldialdehyde, which forms a fluorescent complex with GSH and can be monitored at 350 nm–420 nm.

Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples.

Publisher Summary Glutathione (GSH) is the most important nonprotein thiol in living systems and is of widespread occurrence in the intracellular milieu of animals, plants, and microorganisms GSH was isolated and named by the English biochemist Frederick Gowland Hopkins This chapter discusses GSH status, the biologically relevant chemistry of GSH, the forms in which GSH can be present within the cell, along with the GSH content of cells and the methods for analysis of this substance GSH-related biochemical reactions and the biological roles of GSH are discussed in the chapter The use of perturbations in GSH status as a means for investigating GSH-related phenomena and an analysis of the consequences of perturbation are presented A short summary of genetic lesions related to GSH is also included Like chemically induced perturbations in GSH status, genetic lesions provide valuable insights into the role of GSH in normal functions and processes in cells The chapter concludes with some brief comments about the future of the relationship of GSH status to cellular processes

The Glutathione Status of Cells

Analysis of glutathione: implication in redox and detoxification.

A fluorometric method for determination of oxidized and reduced glutathione in tissues.

Receptors for insulin and estradiol and transport of selected amino acids were examined in two rodent mammary tumor models. Carcinomas induced by 7,12-dimethylbenz(a)anthracene are hormone dependent, and the majority regress in diabetic or ovariectomized hosts; in the former insulin binding was increased and estrogen receptor decreased, whereas in the latter insulin binding was decreased. Insulin treatment of diabetic tumor-bearing animals usually reactivated tumor growth, and this was accompanied by an increased estrogen-binding capacity. The R3230AC mammary adenocarcinoma, a transplantable hormone-responsive tumor, grows faster in diabetic hosts and is inhibited by insulin therapy. Tumors from diabetic animals display higher insulin binding and reduced estrogen receptor. Ovariectomy resulted in a greater than 2-fold elevation in insulin binding in the R3230AC tumor, and estrogen treatment reduced insulin binding. In short-term tissue culture of R3230AC cells, estradiol in vitro appeared to decrease insulin binding and insulin increased estrogen-binding capacity.

Enzymatically dissociated R3230AC tumor cells were used for characterization of carrier-mediated transport of various substrates. Entry of glucose, proline, or phenylalanine demonstrated characteristics similar to those of normal cells. However, with α-aminoisobutyric acid, both Na+-independent and Na+-dependent carrier-mediated entry was observed, suggesting that this analog entered these tumor cells by more than one system. Cells from R3230AC tumors obtained from diabetic hosts showed increased entry of proline or α-aminoisobutyric acid; insulin treatment of diabetic rats returned the rate of entry of these substrates to levels seen in tumor cells from intact rats. Phenylalanine entry was not affected by alteration of the insulin level of the host, indicating that the effects of insulin are specific. Preliminary results showed that estrogen therapy reduced entry of proline and leucine. Thus, entry of proline in vitro could be related to the effects of hormones on growth behavior of the tumor in vivo , and further investigation could provide a better understanding of these hormonal interrelationships at the cellular level.

https://cancerres.aacrjournals.org/content/canres/38/11_Part_2/4076.full.pdf

Regulatory interrelationships for insulin and estrogen action in mammary tumors.

The R3230AC mammary carcinoma grows faster in diabetic rats; tumor cells from diabetic rats demonstrate increased proline transport and incorporation into proteins compared to tumor cells obtained from intact rats. Tumor cells from insulin-treated diabetic rats showed a decreased rate of proline transport and a lower amount of proline incorporation, both of which were comparable to that in cells from intact rats. The effects of insulin in vivo were time and dose related. The rate of α-aminoisobutyric acid entry, but not phenylalanine or leucine, was also higher in tumor cells from diabetic rats than in those from intact rats. Kinetic analysis of α-aminoisobutyric acid uptake indicated that α-aminoisobutyric acid was transported by two carriers. The increase in α-aminoisobutyrate uptake in the absence of insulin was due to an increase (∼170%) in the V max of the Na + -dependent component; no difference in K m was observed. The Na + -independent component of α-aminoisobutyric acid uptake was not significantly increased in cells from diabetic rats. No differences were observed in 3- O -methylglucose transport, 14 CO 2 production from uniformly labeled glucose, or incorporation of labeled glucose into fatty acids for cells from intact or diabetic rats. In contrast, insulin in vitro increased proline transport and its incorporation in cells from diabetic but not from intact rats. The response of cells from insulin-treated diabetic animals was blunted. These responses to insulin in vitro were shown to be dose related and occurred over the physiological range for insulin in vivo . Thus, the enhanced proline transport and incorporation seen in cells from diabetic rats may contribute to the enhanced tumor growth; administration of insulin in vivo reversed this effect on proline and reduced tumor growth. We suggest that these paradoxical effects of insulin in vivo versus those in vitro on proline transport may be due to the presence of other hormones in vivo , which modulate the effects of insulin.

https://cancerres.aacrjournals.org/content/canres/38/11_Part_1/3646.full.pdf

Effects of insulin in vivo and in vitro on amino acid transport into cells from the R3230AC mammary adenocarcinoma and their relationship to tumor growth.

Characteristics of proline transport into R3230AC mammary tumor cells.

The effects of estrogens on transport and incorporation of amino acids into the R3230AC mammary adenocarcinoma were studied in vivo and in vitro . Dissociated tumor cells from ovariectomized rats, like those from diabetic rats, displayed elevated transport of proline, representing entry by the A system; transport of phenylalanine (L system) was unaltered, as was glucose transport and its utilization. Administration of estradiol valerate decreased the entry of proline into tumor cells from intact, diabetic, or ovariectomized animals; the response to the steroid hormone was greater in ovariectomized or diabetic rats compared to intact animals. The time course of the effects of estrogen treatment was examined in diabetic rats. By 72 hr, transport of both proline and leucine was significantly decreased; incorporation of leucine into proteins and uridine into RNA was significantly reduced by 24 hr after injection of estradiol valerate. The effects of estrogen in vivo to reduce transport of amino acids and their incorporation into proteins appeared to correlate with the reduced tumor growth observed. Experiments were performed to examine the effects of 17β-estradiol in vitro on amino acid transport into dissociated cells from ovariectomized or diabetic rats. Under these experimental conditions, 17β-estradiol (10 -6 m) inhibited proline transport with little or no effect on leucine transport in cells from ovariectomized rats; in cells from diabetic rats, proline transport and leucine incorporation were significantly reduced by estradiol, whereas phenylalanine transport was slightly inhibited (∼20%). The effect of estradiol in vitro was also manifest in tumor cells obtained from diabetic rats treated in vivo with estradiol valerate; estradiol in vitro caused a further reduction in proline transport but not in leucine transport, results that imply some specificity to the action of estrogen on the A system. Since we had earlier shown that insulin action on transport in these tumor cells was directed towards the A system, we examined the effects of insulin, estradiol, and their combination in vitro on proline and leucine transport. Insulin (10 -8 m) stimulated proline transport; 17β-estradiol, at a selected lower level of 10 -8 m, inhibited proline transport. When both were added in vitro , estradiol (10 -8 m) was capable of significantly reducing the insulin (10 -8 m)-induced increase in proline transport. Leucine transport was not altered in any of these experiments. Together, these data suggest that estrogens are capable of inhibiting amino acid transport into the R3230AC mammary carcinoma, an effect that is compatible with reduced tumor growth. The possible relationship of estrogen and insulin at the level of amino acid transport remains to be elucidated.

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Paul J. Hissin

Papers

A fluorometric method for determination of oxidized and reduced glutathione in tissues.

Regulatory interrelationships for insulin and estrogen action in mammary tumors.

Effects of insulin in vivo and in vitro on amino acid transport into cells from the R3230AC mammary adenocarcinoma and their relationship to tumor growth.

Characteristics of proline transport into R3230AC mammary tumor cells.

Effects of estrogen to alter amino acid transport in R3230AC mammary carcinomas and its relationship to insulin action.