BACKGROUND
clinical and molecular characteristics of tumors may assist in the development of more efficient and less toxic therapeutic strategies.
in this way, the expression of antioxidant proteins in tumor cells has been investigated as a predictor and prognostic factor in cytotoxic treatment in breast cancer  <cit> .
when there is an excessive production of reactive oxidative species , the antioxidant defense system is triggered, which is of great importance in the physiopathology of diverse cancer types, including breast cancer  <cit> .
the consequence of this process is the loss of cellular function and progression towards cell death.
moreover, oxidative stress leads to dna damage and mutations in tumor suppressor genes, events that can be important in the initiation of carcinogenesis  <cit> .
gsh is a tripeptide of glycine, cysteine and glutamic acid that is found in high concentration intracellularly, being the most abundant low molecular mass thiol  <cit> .
synthesis of gsh requires the consecutive action of two enzymes, first glutamate-cysteine ligase  that conjugates glutamic acid and cysteine forming gama-glutamyl cysteine.
this compound containing cysteine with a sulfhydryl group  is responsible for the antioxidant activity of gsh.
the second reaction is the binding of gama-glutamyl cysteine with glycine by the enzyme glutathione synthetase , giving rise to the tripeptide gama-glutamyl cysteine glycine-glutathione  <cit> .
gsh-px is an  <dig> kda protein composed of four identical sub-units.
five isoforms of gsh-px are well characterized in mammals and show tissue-specific distribution.
alteration of these enzyme levels are associated with diverse cancer types, including skin, kidney, intestine and breast cancer  <cit> .
gsts are a family of intracellular enzymes of detoxification phase ii that catalyze diverse electrophilic compounds conjunction to gsh, blocking dna mutations of the cells  <cit> .
admittedly, the classification of mammary neoplasms in female dogs is complex and detailed, but it cannot predict the progress of the illness and the survivor time of the animal with cancer nor the chemotherapeutic and/or hormonal treatment feedback.
nowadays, using the possibility of immunohistochemistry diagnosis of the tumors by biomarkers, prognosis can be more accurate and its predictable value can also direct genetic treatment.
besides, the biomarkers that apparently do not have independent prognostic value, together with the clinic-pathologic parameters, may assist in the evaluation of feedback to the patient’s clinical development.
despite advances in identification of genes involved in tumor growth, progression and resistance to drugs, the way these genes interact with one another remains unclear.
therefore a better understanding of how molecular markers are associated with therapeutic response is indispensable in selecting the correct drugs for treatment.
the capacity to establish primary cell cultures of tumors is an important requirement in cancer research, allowing the identification of important prognostic factors and therapeutic agents.
moreover, this is the best way of assessing the predictive value of adjuvant treatment that might also be given to cancer patients  <cit> .
primary cultures have been successfully used to get answers to drug dosages and to support new anti-cancer drug development  <cit> .
the enzymatic system of glutathione/glutathione transferases  is seen as one of the most important in cellular resistance to multiple drugs , involving the alteration of gsh level, the expression of genes that codify the enzymes responsible for its synthesis, and the genes that code for gsts  <cit> .
the aim of this study has been to assess the expression of gsh and the enzymes gstpi and gsh-px by immunohistochemistry in mammary tumors and to infer their prognostic value.
an in vitro study of molecular analyses was made, verifying the resistance of neoplastic cells to the chemotherapeutic agent.
RESULTS
clinical data
bitches from the test group were evaluated with respect to physical , pathological  and clinical  characteristics.
the animals ranged from  <dig> to  <dig> years of age, the mean at diagnosis being  <dig> years.
with respect to the breed, there were  <dig>  mongrels, eight  poodles, three  cockers and three  boxers, two  pinchers, one  basset and one  dachshund.
a total of  <dig> canine mammary lesions  were histopathologically diagnosed, the benign tumors being composed of three mixed benign tumors, two sarcomatoid-like lesion and one papilloma while the malignant tumors included  <dig> carcinomas in mixed tumors, three papillary carcinomas, two carcinosarcomas, two tubulopapillary carcinomas, one comedocarcinoma, one solid carcinoma and one inflammatory carcinoma.
there was a predominance of clinical staging i .
among the clinicopathological characteristics,  <dig>  of the tumors had multiple numbers of nodules,  <dig>  tumors had the absence of ulceration and  <dig>  had moderate vascularization.
the metastasis and death index was 27% where all the cases of death occurred from pulmonary metastasis.
all the data were described in table  <dig>
*significant value to student’s t test # anova test.
immunohistochemistry study
immunostaining of gsh was evident in the cytoplasm of neoplastic cells, and gstpi and gsh-px were also focally seen in the nuclei.
both antibodies labeled the stroma .
there was no correlation between the expression of the gsh and clinical characteristics and pathology with the age, time course, histological type, clinical staging, number of nodules and tumor vascularization .
however, the high expression of gsh had statistically significant correlation in female dogs with non-ulcerated tumors .
the expression of gsh, gstpi and gsh-px by immunohistochemistry was compared with the clinical development of the female dogs, including metastasis and death.
the metastasis index was  <dig> % and the same for the death index .
gsh expression increased in female dogs that survived to the end of follow-up  and it also increased in those female dogs that did not present metastasis .
there was no significant statistical relation between the analyzed variables and the expression of the enzymes gstpi and gsh-px .
to generate the roc curve, the values of the gsh, gstpi and gsh-px expression of the female dogs that died were compared to those that survived.
the roc curves indicate the performance and the limit values for gsh, gstpi and gsh-px expression in predicting the risk of mortality, and calculated the sensitivity/specificity, thereby establishing the best cut-off to discriminate high-death-risk dogs, which for gsh expression was: m.d.o.
=  <dig> u.a.
, gstpi expression was: m.d.o.
=  <dig> u.a.
and gsh-px expression: m.d.o.
<dig> u.a.
.
the dogs were followed-up from 1– <dig> months for overall survival, with a median of  <dig> days.
the relationship between gsh, gsh-px and gstpi enzyme expression and survival of patients was evaluated by kaplan–meier analysis and log-rank test, using the cut-off value established by the roc curve.
we found that the overall survival of patients with low gsh expression was significantly shorter than patients with high gsh expression .
the median survival time of dogs that died was  <dig>  months.
there was no correlation between gstpi and gsh-px expression and overall survival .
in vitro study
<dig> tumor specimens were used from the  <dig> samples used for immunohistochemistry, which were histopathologically identified as seven carcinomas in mixed tumors, two tubulopapillary carcinomas and one papillary carcinoma.
after treatment with doxorubicin, all the samples showed underexpression of the gsh gene compared with the control group   and the gss gene was also expressed more weakly in the presence of the drug in 90% of the samples  .
there was no significant statistical difference between gsh-px gene expression in the control group relative to the group treated with doxorubicin .
discussion
in this study, there was an increase in gsh expression in tumors without ulceration, not metastatic tumors and low mortality.
our results are in agreement with buser et al.
<cit>  who demonstrated that high levels of gsh, gstpi and gsh-px were associated with favorable clinical characteristics and good prognosis, whereas low levels of gsh and gst activity were associated with more aggressive or more advanced disease.
after  <dig> months, no significant results were observed when comparing the gstpi and gsh-px expression with metastasis and death in our dogs.
our results are in agreement with jardim et al.
<cit>  where no statistical correlation was found between gstpi expression and local recurrence and/or metastasis either.
in this study, we verified the influence of chemotherapy with doxorubicin on the gsh and gsh-px genes expression.
according to cell culture growing curves, doxorubicin efficiently causes tumor cell death.
moreover, doxorubicin treatment reduces the expression of the genes gclc and gss, respectively, in treated tumor cells over untreated tumor cells.
gclc sensitizes the enzyme glutamate cysteine ligase to catalyze the first step for gsh synthesis and gss codes for glutathione synthetase catalyzing the second and last step of synthesis of gsh.
together, these data support the notion that doxorubicin reduces a pattern of gene expression associated with modulation of the antioxidant capacity of mammary tumor cells.
many studies have shown alterations in gsh activity after in vitro treatment with doxorubicin or similar drugs.
our results agree with ozkan and fiskin  <cit> , in which epirubicin - a structural analog of doxorubicin - decreases gsh activity after  <dig> h in cultured mammary neoplastic cells.
some studies suggest that reactive oxygen species  production in doxorubicin treatment is responsible for cytotoxicity in neoplastic cells, which could induce the synthesis of antioxidant enzymes, such as gsh and gsh-px, making the neoplastic cells resistant to oxidative damage  <cit> .
in the same way, han et al.
<cit>  found greater sensitivity to doxorubicin when the gsh levels decreased in the mammary carcinoma cell line .
however, in contrast, di et al.
<cit>  demonstrated that gsh overexpression did not prevent tumor cell apoptosis after treatment with doxorubicin, suggesting that the cytotoxicity of the drug might not be wholly related to ros production.
additionally, the involvement of gsh in the therapeutic response to doxorubicin is controversial.
some authors suggest that gsh inhibition leads to the drug accumulating in the nucleus of the tumor cells  <cit> , whereas others suggest that resistance to doxorubicin and other similar drugs is not directly related to the binding of the drug to gsh  <cit> .
exactly what intracellular alterations are responsible for the mdr phenotype in neoplastic cells remain largely unknown.
there is a suggestion that high concentrations of gsh, gsh-px and gstpi, independent of other intracellular alterations, do not decisively contribute to mdr  <cit> .
the resistance to the antineoplastic agent cisplatin, for instance, has been associated to the high expression of the enzyme gama glutamyl transpeptidase  localized at the cell membrane, which is responsible for its degradation.
thereby, despite the fact that cytotoxic treatment decreases intracellular gsh concentration, this enzyme hydrolyzes serum gsh, releasing the necessary amino acids for intracellular gsh replenishment  <cit> .
the enzyme ggt is mainly found in liver and kidney cells; however, resistance to drugs due to high levels of this enzyme has been found in many cancer types including some mammary tumors  <cit> .
in this context, there are many studies trying to identify agents that can increase the efficiency of conventional drugs and/or reverse the mdr phenotype in neoplastic cells  <cit> .
despite the correlation between high expression of these genes and the mdr, some studies suggest that using agents that increase gsh, gsh-px and gsts levels during drug treatment is a promising way to reduce oxidative stress, thus reducing the collateral effects of chemotherapy in breast cancer patients  <cit> .
the mammary tumors cultivated in vitro showed underexpression of the gsh coding genes and no alteration to the expression of gsh-px, after treatment with doxorubicin.
this can be explained by the interaction between these two enzymes in the antioxidant defense of tumor cells.
gsh-px has selenium at its catalytic site and uses gsh as an electron donor for the reduction of h2o <dig> to h2o  <cit> .
so the results show that gsh and gsh-px don’t provide adequate defense against oxidants, rendering them sensitive to drugs.
in this context, it is necessary to clarify the mechanism of these enzymes in cell resistance and their true prognostic value  <cit> .
knowledge of gsh expression and its enzymes in a wide range of biology processes is expanding.
advances in analytical techniques and protein localization will present many opportunities for the development of therapeutic interventions in cancer and other diseases related to levels of oxidative stress  <cit> .
CONCLUSIONS
high expression of gsh in neoplastic mammary cells from female dogs is associated with the best clinical outcome, including a greater survival time.
therefore this high expression plays an important role in the clinical outcome of patients with mammary tumors and can be used as prognostic marker for mammary neoplasms in female dogs.
treatment of mammary neoplastic cells with doxorubicin in vitro is an efficient way to induce cell death, reducing the expression of the gclc and gss genes that are related to antioxidant production.
thus they can be considered potential predictive markers on the therapeutic feedback to breast cancer.
more studies, using large cohorts in future studies, are needed to confirm the prognostic value of these markers in the mammary tumor progression in female dogs.

 clinical and molecular characteristics of tumors may assist in the development of more efficient and less toxic therapeutic strategies. in this way, the expression of antioxidant proteins in tumor cells has been investigated as a predictor and prognostic factor in cytotoxic treatment in breast cancer. when there is an excessive production of reactive oxidative species, the antioxidant defense system is triggered, which is of great importance in the physiopathology of diverse cancer types, including breast cancer. the consequence of this process is the loss of cellular function and progression towards cell death. moreover, oxidative stress leads to dna damage and mutations in tumor suppressor genes, events that can be important in the initiation of carcinogenesis. gsh is a tripeptide of glycine, cysteine and glutamic acid that is found in high concentration intracellularly, being the most abundant low molecular mass thiol. synthesis of gsh requires the consecutive action of two enzymes, first glutamate-cysteine ligase that conjugates glutamic acid and cysteine forming gama-glutamyl cysteine. this compound containing cysteine with a sulfhydryl group is responsible for the antioxidant activity of gsh. the second reaction is the binding of gama-glutamyl cysteine with glycine by the enzyme glutathione synthetase, giving rise to the tripeptide gama-glutamyl cysteine glycine-glutathione. gsh-px is an kda protein composed of four identical sub-units. five isoforms of gsh-px are well characterized in mammals and show tissue-specific distribution. alteration of these enzyme levels are associated with diverse cancer types, including skin, kidney, intestine and breast cancer. gsts are a family of intracellular enzymes of detoxification phase ii that catalyze diverse electrophilic compounds conjunction to gsh, blocking dna mutations of the cells. admittedly, the classification of mammary neoplasms in female dogs is complex and detailed, but it cannot predict the progress of the illness and the survivor time of the animal with cancer nor the chemotherapeutic and/or hormonal treatment feedback. nowadays, using the possibility of immunohistochemistry diagnosis of the tumors by biomarkers, prognosis can be more accurate and its predictable value can also direct genetic treatment. besides, the biomarkers that apparently do not have independent prognostic value, together with the clinic-pathologic parameters, may assist in the evaluation of feedback to the patient’s clinical development. despite advances in identification of genes involved in tumor growth, progression and resistance to drugs, the way these genes interact with one another remains unclear. therefore a better understanding of how molecular markers are associated with therapeutic response is indispensable in selecting the correct drugs for treatment. the capacity to establish primary cell cultures of tumors is an important requirement in cancer research, allowing the identification of important prognostic factors and therapeutic agents. moreover, this is the best way of assessing the predictive value of adjuvant treatment that might also be given to cancer patients. primary cultures have been successfully used to get answers to drug dosages and to support new anti-cancer drug development. the enzymatic system of glutathione/glutathione transferases is seen as one of the most important in cellular resistance to multiple drugs, involving the alteration of gsh level, the expression of genes that codify the enzymes responsible for its synthesis, and the genes that code for gsts. the aim of this study has been to assess the expression of gsh and the enzymes gstpi and gsh-px by immunohistochemistry in mammary tumors and to infer their prognostic value. an in vitro study of molecular analyses was made, verifying the resistance of neoplastic cells to the chemotherapeutic agent.  clinical data bitches from the test group were evaluated with respect to physical, pathological and clinical characteristics. the animals ranged from to years of age, the mean at diagnosis being years. with respect to the breed, there were mongrels, eight poodles, three cockers and three boxers, two pinchers, one basset and one dachshund. a total of canine mammary lesions were histopathologically diagnosed, the benign tumors being composed of three mixed benign tumors, two sarcomatoid-like lesion and one papilloma while the malignant tumors included carcinomas in mixed tumors, three papillary carcinomas, two carcinosarcomas, two tubulopapillary carcinomas, one comedocarcinoma, one solid carcinoma and one inflammatory carcinoma. there was a predominance of clinical staging i. among the clinicopathological characteristics, of the tumors had multiple numbers of nodules, tumors had the absence of ulceration and had moderate vascularization. the metastasis and death index was 27% where all the cases of death occurred from pulmonary metastasis. all the data were described in table *significant value to student’s t test # anova test. immunohistochemistry study immunostaining of gsh was evident in the cytoplasm of neoplastic cells, and gstpi and gsh-px were also focally seen in the nuclei. both antibodies labeled the stroma. there was no correlation between the expression of the gsh and clinical characteristics and pathology with the age, time course, histological type, clinical staging, number of nodules and tumor vascularization. however, the high expression of gsh had statistically significant correlation in female dogs with non-ulcerated tumors. the expression of gsh, gstpi and gsh-px by immunohistochemistry was compared with the clinical development of the female dogs, including metastasis and death. the metastasis index was % and the same for the death index. gsh expression increased in female dogs that survived to the end of follow-up and it also increased in those female dogs that did not present metastasis. there was no significant statistical relation between the analyzed variables and the expression of the enzymes gstpi and gsh-px. to generate the roc curve, the values of the gsh, gstpi and gsh-px expression of the female dogs that died were compared to those that survived. the roc curves indicate the performance and the limit values for gsh, gstpi and gsh-px expression in predicting the risk of mortality, and calculated the sensitivity/specificity, thereby establishing the best cut-off to discriminate high-death-risk dogs, which for gsh expression was: m.d.o. = u.a., gstpi expression was: m.d.o. =  u.a. and gsh-px expression: m.d.o. u.a.. the dogs were followed-up from 1– months for overall survival, with a median of days. the relationship between gsh, gsh-px and gstpi enzyme expression and survival of patients was evaluated by kaplan–meier analysis and log-rank test, using the cut-off value established by the roc curve. we found that the overall survival of patients with low gsh expression was significantly shorter than patients with high gsh expression. the median survival time of dogs that died was months. there was no correlation between gstpi and gsh-px expression and overall survival. in vitro study tumor specimens were used from the samples used for immunohistochemistry, which were histopathologically identified as seven carcinomas in mixed tumors, two tubulopapillary carcinomas and one papillary carcinoma. after treatment with doxorubicin, all the samples showed underexpression of the gsh gene compared with the control group and the gss gene was also expressed more weakly in the presence of the drug in 90% of the samples. there was no significant statistical difference between gsh-px gene expression in the control group relative to the group treated with doxorubicin. discussion in this study, there was an increase in gsh expression in tumors without ulceration, not metastatic tumors and low mortality. our results are in agreement with buser et al. who demonstrated that high levels of gsh, gstpi and gsh-px were associated with favorable clinical characteristics and good prognosis, whereas low levels of gsh and gst activity were associated with more aggressive or more advanced disease. after months, no significant results were observed when comparing the gstpi and gsh-px expression with metastasis and death in our dogs. our results are in agreement with jardim et al. where no statistical correlation was found between gstpi expression and local recurrence and/or metastasis either. in this study, we verified the influence of chemotherapy with doxorubicin on the gsh and gsh-px genes expression. according to cell culture growing curves, doxorubicin efficiently causes tumor cell death. moreover, doxorubicin treatment reduces the expression of the genes gclc and gss, respectively, in treated tumor cells over untreated tumor cells. gclc sensitizes the enzyme glutamate cysteine ligase to catalyze the first step for gsh synthesis and gss codes for glutathione synthetase catalyzing the second and last step of synthesis of gsh. together, these data support the notion that doxorubicin reduces a pattern of gene expression associated with modulation of the antioxidant capacity of mammary tumor cells. many studies have shown alterations in gsh activity after in vitro treatment with doxorubicin or similar drugs. our results agree with ozkan and fiskin, in which epirubicin - a structural analog of doxorubicin - decreases gsh activity after h in cultured mammary neoplastic cells. some studies suggest that reactive oxygen species production in doxorubicin treatment is responsible for cytotoxicity in neoplastic cells, which could induce the synthesis of antioxidant enzymes, such as gsh and gsh-px, making the neoplastic cells resistant to oxidative damage. in the same way, han et al. found greater sensitivity to doxorubicin when the gsh levels decreased in the mammary carcinoma cell line. however, in contrast, di et al. demonstrated that gsh overexpression did not prevent tumor cell apoptosis after treatment with doxorubicin, suggesting that the cytotoxicity of the drug might not be wholly related to ros production. additionally, the involvement of gsh in the therapeutic response to doxorubicin is controversial. some authors suggest that gsh inhibition leads to the drug accumulating in the nucleus of the tumor cells, whereas others suggest that resistance to doxorubicin and other similar drugs is not directly related to the binding of the drug to gsh. exactly what intracellular alterations are responsible for the mdr phenotype in neoplastic cells remain largely unknown. there is a suggestion that high concentrations of gsh, gsh-px and gstpi, independent of other intracellular alterations, do not decisively contribute to mdr. the resistance to the antineoplastic agent cisplatin, for instance, has been associated to the high expression of the enzyme gama glutamyl transpeptidase localized at the cell membrane, which is responsible for its degradation. thereby, despite the fact that cytotoxic treatment decreases intracellular gsh concentration, this enzyme hydrolyzes serum gsh, releasing the necessary amino acids for intracellular gsh replenishment. the enzyme ggt is mainly found in liver and kidney cells; however, resistance to drugs due to high levels of this enzyme has been found in many cancer types including some mammary tumors. in this context, there are many studies trying to identify agents that can increase the efficiency of conventional drugs and/or reverse the mdr phenotype in neoplastic cells. despite the correlation between high expression of these genes and the mdr, some studies suggest that using agents that increase gsh, gsh-px and gsts levels during drug treatment is a promising way to reduce oxidative stress, thus reducing the collateral effects of chemotherapy in breast cancer patients. the mammary tumors cultivated in vitro showed underexpression of the gsh coding genes and no alteration to the expression of gsh-px, after treatment with doxorubicin. this can be explained by the interaction between these two enzymes in the antioxidant defense of tumor cells. gsh-px has selenium at its catalytic site and uses gsh as an electron donor for the reduction of h2o to h2o. so the results show that gsh and gsh-px don’t provide adequate defense against oxidants, rendering them sensitive to drugs. in this context, it is necessary to clarify the mechanism of these enzymes in cell resistance and their true prognostic value. knowledge of gsh expression and its enzymes in a wide range of biology processes is expanding. advances in analytical techniques and protein localization will present many opportunities for the development of therapeutic interventions in cancer and other diseases related to levels of oxidative stress.  high expression of gsh in neoplastic mammary cells from female dogs is associated with the best clinical outcome, including a greater survival time. therefore this high expression plays an important role in the clinical outcome of patients with mammary tumors and can be used as prognostic marker for mammary neoplasms in female dogs. treatment of mammary neoplastic cells with doxorubicin in vitro is an efficient way to induce cell death, reducing the expression of the gclc and gss genes that are related to antioxidant production. thus they can be considered potential predictive markers on the therapeutic feedback to breast cancer. more studies, using large cohorts in future studies, are needed to confirm the prognostic value of these markers in the mammary tumor progression in female dogs. 
this study evaluated the expression of gsh and the gsh-px and gstpi enzymes by immunohistochemistry in canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. the analysis of the relative expression of genes responsible for the synthesis of gsh and gsh-px by quantitative pcr was done with cultured cells of tumor fragments from dogs with mammary tumors. therefore, high expression of the gsh seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. there was significant correlation of survival with the increase of gsh. the expression of the gsh-px and gstpi enzymes showed no statistically significant correlation with the analyzed variables. the in vitro doxorubicin treatment significantly reduces the expression of gclc and gss genes so we can consider them to be candidates for predictive markers of therapeutic response in mammary cancer. glutathione is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase and glutathione s transferase pi, it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. in an in vitro study, the expression of the genes glutamate cysteine ligase and glutathione synthetase that synthesize gsh and gsh-px gene were verified by qpcr and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy. the results showed that high immunoexpression of gsh was correlated to the absence of tumor ulceration and was present in dogs without metastasis. the immunohistochemical expression of gsh, gsh-px and gstpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death. high gsh immunoexpression was associated with better clinical outcomes.
 glutathione is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase and glutathione s transferase pi, it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. this study evaluated the expression of gsh and the gsh-px and gstpi enzymes by immunohistochemistry in canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. in an in vitro study, the expression of the genes glutamate cysteine ligase and glutathione synthetase that synthesize gsh and gsh-px gene were verified by qpcr and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy.  the immunohistochemical expression of gsh, gsh-px and gstpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death. the results showed that high immunoexpression of gsh was correlated to the absence of tumor ulceration and was present in dogs without metastasis. there was significant correlation of survival with the increase of gsh. the expression of the gsh-px and gstpi enzymes showed no statistically significant correlation with the analyzed variables. the analysis of the relative expression of genes responsible for the synthesis of gsh and gsh-px by quantitative pcr was done with cultured cells of tumor fragments from dogs with mammary tumors. the culture cells showed a decrease in gclc and gss expression when compared with no treated cells. high gsh immunoexpression was associated with better clinical outcomes.  therefore, high expression of the gsh seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. the in vitro doxorubicin treatment significantly reduces the expression of gclc and gss genes so we can consider them to be candidates for predictive markers of therapeutic response in mammary cancer. mammary neoplasiaglutathioneimmunohistochemistryreal-time pcroxidative stress 