Introduction
Glutathione is an endogenous tripeptide composed of glutamine, cysteine, and glycine. It is a potent anti-oxidant with numerous functions, including protecting cells from oxidative damage, detoxification, and maintaining immune function.1,2,3 Glutathione is also involved in a variety of cellular functions, including DNA repair, cell cycle control, cell signaling, and transcription factor regulation.2 Low tissue concentrations of GSH result in an overall increase in ROS and secondary oxidative stress, a process that has been associated with numerous disease processes, including carcinogenesis.1,4
Reduction-oxidation homeostasis has an important role in carcinogenesis, including both tumor progression and tumor suppression. Reactive oxygen species cause direct damage to DNA and induce genomic instability, which is a critical step in tumor initiation and progression.4 Reactive oxygen species are also believed to have a role in the cellular processes associated with tumor metastasis, including loss of cell-cell adhesions, cell survival after matrix detachment, and cell migration. Additionally, ROS can stimulate tumor proliferation through activation of mitogenic pathways.4 Intracellular antioxidants are directly related to the concentration of ROS and play a critical role in redox homeostasis.3 When the intracellular ROS concentration increases, the cell triggers activation of gene transcription, resulting in increased production of antioxidants.4 However, this system can be overwhelmed by an extreme ROS concentration, resulting in cellular and DNA damage.3,4 Glutathione has an important role in eliminating cellular carcinogens and may prevent or slow the development of some cancers.3
In addition to the role redox homeostasis has in carcinogenesis, GSH also plays a functional role in conventional antineoplastic treatments, including radiation therapy and chemotherapy. These treatments use ROS production to damage or induce cancer cell death.4 However, this ROS production is not tumor-cell specific, and ROS within healthy cells contribute to undesirable chemotherapy and radiation toxic effects. These toxic effects can have considerable impacts on treatment tolerance and patient quality of life.5 Supplementation with GSH and its precursors has been shown in multiple studies5,6,7,8,9 to reduce chemotherapy-related toxic effects, without an adverse impact on treatment efficacy. Despite these findings, concurrent antioxidant supplementation and conventional antineoplastic treatment remains controversial and current recommendations are poorly defined.5
The complexities of the cellular redox systems are only beginning to be understood. Current understanding of how ROS and antioxidants contribute to or protect against tumorigenesis is limited, as is our understanding of how to safely manipulate this system to enhance anticancer treatments and improve patient quality of life. To date, most studies involving in vivo manipulation of the tumor-host redox balance have been performed in rodents. These studies often involve the use of tumor xenografts in immunocompromised rodents, and they fail to accurately reproduce the environmental risk factors, host genetic variation, natural tumor biology, and tumor-immune system interactions that are crucial to understanding and treating naturally occurring human cancers.6 Dogs are increasingly gaining attention as subjects for investigation of human cancers, which would overcome some of the limitations associated with in vitro and preclinical rodent studies.10,11 Dogs often occupy the same environment as people do and are often inadvertently exposed to the same carcinogens. Interestingly, dogs can develop spontaneous tumors with histopathologic characteristics similar to those of human tumors, with similar patterns of tumor evolution, recurrence, and metastasis. Dogs also have intact immune systems when healthy and varied genetic backgrounds, which are often lacking in rodent-based populations.10,11 When considering dogs as subjects for evaluating the role of antioxidant supplementation in the development, progression, and treatment of cancer, their shorter life span and more rapid disease progression relative to humans would allow for prompt assessment of both therapeutic and chemoprevention studies.12 Additionally, owners are often motivated to enroll their pets in clinical trials to offset the cost of cancer treatment and are generally quite focused on improving or maintaining their dog's quality and quantity of life.12
Before endogenous and supplemental GSH is evaluated in dogs with cancer, it would be important to measure cellular GSH concentrations and evaluate the safety and efficacy of GSH supplementation in healthy dogs. Glutathione has variable oral bioavail-ability and is not readily taken up by most cells.13 Oral supplementation with l-cysteine, the rate-limiting substrate in GSH biosynthesis, can increase intracellular GSH concentrations but has neurotoxic and mutagenic effects at therapeutic doses.14 The l-cysteine prodrug RibCys results in slow intracellular release of l-cysteine and increased intracellular GSH concentrations in laboratory animals.14,15 Previous studies of RibCys administration in mice,14,15 swine,16 and humans have demonstrated no toxic effects.17 Our goal was to evaluate the safety of orally administering a RibCys supplement to healthy dogs and the effect of this supplementation on erythrocyte GSH concentration. We hypothesized that administration of the RibCys supplement would result in no measurable clinical or biochemical toxic effects and would cause an increase in erythrocyte GSH concentration.
Materials and Methods
Animals
Twenty-four healthy adult dogs owned by employees of the Animal Medical Center, New York City, were enrolled in the study. Dogs were deemed healthy on the basis of clinical and owner history, physical examination, and routine laboratory testing, including a CBC, serum biochemical analysis, and urinalysis. For inclusion in the study, dogs were required to weigh at least 15 kg and be at least 2 years of age at the time of enrollment. Dogs that had received any dietary supplement products during the period 1 month prior to enrollment were excluded. The study protocol was approved by the Animal Medical Center's Institutional Animal Care and Use Committee. All owners provided informed consent before enrollment of their dogs in the study.
Study design and treatments
A randomized, double-blinded, controlled trial was performed. Dogs were randomly assigned by means of a random number generator to a RibCys or control group (n = 12/group). Dogs in the RibCys group received a commercially available RibCys supplement (two 250-mg capsules,a PO, q 12 h for 4 weeks). Dogs in the control group received the cellulose vehicle for RibCys as a placebo (2 capsules,a PO, q 12 h for 4 weeks). The RibCys and placebo capsules were identical in appearance. The investigators and owners were blinded to the treatment group assignment for the duration of the clinical trial and data analysis.
On days 0 (the day before treatment began), 7, 14, 21, and 28, all dogs underwent a physical examination and owners were asked to complete a questionnaire on study medication administration, quality of life, and adverse effects over the prior 7 days (Supplementary Appendices S1 and S2, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.8.653). Pill counts were performed to ensure owner compliance in administration. On days 0, 7, 14, 21, and 28 after food had been withheld from dogs for 12 hours and before the next dose of RibCys supplement or placebo was administered, blood samples were collected by jugular venipuncture into evacuated plastic tubes containing EDTA and plain evacuated tubes for CBC and serum biochemical analysis, respectively, and evacuated plastic tubes containing EDTA for erythrocyte GSH assay. At the same time points, a urine sample was collected via cystocentesis for urinalysis. Blood samples for GSH assay were immediately placed in an ice bath pending analysis. The remaining samples were submitted to a reference laboratoryb for analysis by means of standard methods. Manual review of a blood smear preparation was performed for each CBC sample.
Erythrocyte GSH assay
Within 15 minutes after collection, blood samples for measurement of erythrocyte GSH concentration were centrifuged at 500 × g for 10 minutes. Plasma was harvested and transferred to a plastic vial. This vial and the RBCs remaining in the original tube were frozen at –80°C. On completion of the study, all frozen samples were shipped overnight on dry ice to a university laboratoryc for batch analysis of erythrocyte GSH concentration as described elsewhere.1 For statistical analysis, erythrocyte GSH concentrations were corrected for dilution.
Statistical analysis
One-way ANOVA was performed to compare age and body weight between groups. The effects of a dog's sex on erythrocyte GSH concentration and of treatment on erythrocyte GSH concentration were estimated by means of repeated-measures linear mixed-effects modeling.d,e Our set of candidate models for the effect of treatment on erythrocyte GSH concentration contained 4 models, including a null model (constant), a model differentiating the RibCys and placebo groups (group), an additive model containing group and day of study (group + day), and an interactive model containing the interaction between group and study day (group X day). We fit relatively simple models both because they were capable of addressing our primary research question concerning treatment differences and because of the small sample size. Treatment group was included as a binary covariate and study day as a continuous variate; both were treated as fixed effects. Individual variation in GSH responses was accounted for by treating individual dogs as a random effect in all models. Model assumptions were confirmed, including normality of residuals. Models were compared by use of model weights and the Akaike information criterion corrected for small sample size to guard against selecting overparameterized models. Values of P < 0.05 were considered significant.
Results
Animals
All dogs in the RibCys group (6 spayed females, 5 castrated males, and 1 sexually intact female) and placebo group (5 spayed females, 5 castrated males, and 2 sexually intact females) fully completed the study, with no missed treatments. Mean (range) values for body weight were 26.4 kg (16.1 to 40.3 kg) and 27.8 kg (19.7 to 46.0), respectively, and for age were 7.4 years (2.2 to 11 years) and 5.1 years (2 to 13.7 years). Sex distributions were equivalent between groups, and no significant difference in body weight (P = 0.70) or age (P = 0.09) was found. Overall, dogs included 10 mixed breeds, 4 American Staffordshire Terriers, 2 Labrador Retrievers, 2 Irish Setters, and 1 each of Golden Retriever, Portuguese Water Dog, Border Collie, Cocker Spaniel, German Shepherd Dog, and Carolina Dog.
Three dogs in the RibCys group and 4 in the placebo group had a previous diagnosis of osteoarthritis and were not receiving any supplements or medications at the time of enrollment. Three dogs in the RibCys group and 2 in the placebo group had histories and mild signs of allergic skin disease, which was not being treated at enrollment. One dog in the RibCys group had well-controlled hypoadrenocorticism, and 1 dog in the placebo group had well-controlled hypothyroidism.
Clinical safety
No severe or life-threatening adverse events were reported for dogs in either group during the study period. Minor adverse effects of diarrhea, decreased appetite, and transient mild anemia occurred in both the RibCys group and placebo group. Specifically, 3 and 2 dogs in the RibCys group and placebo group, respectively, had diarrhea; 2 and 1 dog, respectively, had a single day of decreased appetite during the first week of drug administration; and 3 and 2 dogs, respectively, developed a mild transient anemia, with reported Hct values between 36% and 38% (reference interval, 38.3% to 56.5%).
Otherwise, a few other adverse effects were noted during the study period, all of which pertained to dogs in the placebo group. Two dogs in the placebo group had signs of progressive osteoarthritis throughout the study. One dog developed a sudden lameness of the right hind limb during the second week of the study. The lameness resolved with NSAID and doxycycline administration; however, immune-mediated polyarthropathy was diagnosed several months later when the signs recurred. One dog had a mild neutropenia (2.69 × 103 neutrophils/μL; reference interval, 2.94 × 103 to 12.67 × 103 neutrophils/μL) documented at week 4. Another dog had bacteriuria (> 40 bacteria/hpf) identified on urinalysis at the final day of the study. No significant changes from day 0 in serum biochemical variables were identified in either group.
As reported in completed owner questionnaires, several dogs in the RibCys group and no dogs in the placebo group had clinical improvements. From day 7 to the end of the study, 3 dogs in the RibCys group had improved energy and fewer clinical signs of their osteoarthritis. Additionally, 2 dogs had improvement in the health of their skin and coat, which was first noted on day 14 and persisted to the end of the study. For 1 dog with a history of allergic dermatitis, the owner reported a reduction in pruritus and a healthier-appearing coat. The second dog had an area of chronic alopecia that resolved, with normal hair regrowth noted. One dog in the RibCys group had an improvement in appetite, noted on day 14.
Erythrocyte GSH concentrations
Erythrocyte GSH concentration corrected for dilution ranged from 0.90 to 3.36mM over the study period, with an overall mean ± SD value of 2.01 ± 0.41mM (Figure 1). After individual variability was accounted for, erythrocyte GSH concentrations did not differ significantly by sex (β = 0.20; 95% CI, –0.10 to 0.50; P = 0.18), but males had a slightly higher mean value (2.13mM) than did females (1.93mM; Figure 2). Given the 2.5% and 97.5% quantiles from the raw data, normal erythrocyte GSH concentrations ranged from 1.33 to 2.97mM across both sexes.
Erythrocyte GSH concentrations in individual healthy adult dogs the day before (day 0) and at various points during oral administration of 500 mg of a RibCys supplement or placebo (n = 12 dogs/group), PO, every 12 hours for 4 weeks. Dashed lines correspond to females, and solid lines correspond to males.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.653
Erythrocyte GSH concentrations in individual healthy adult dogs the day before (day 0) and at various points during oral administration of 500 mg of a RibCys supplement or placebo (n = 12 dogs/group), PO, every 12 hours for 4 weeks. Dashed lines correspond to females, and solid lines correspond to males.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.653
Erythrocyte GSH concentrations in individual healthy adult dogs the day before (day 0) and at various points during oral administration of 500 mg of a RibCys supplement or placebo (n = 12 dogs/group), PO, every 12 hours for 4 weeks. Dashed lines correspond to females, and solid lines correspond to males.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.653
Erythrocyte GSH concentrations for male (n = 10) and female (14) dogs in both treatment groups over the 28-day study period. Data points represent the raw data, which include repeated measurements for each dog. Mean values (top of gray-shaded bars) and 95% CIs (error bars), derived from a simple linear model of the effect of sex on GSH concentration, are also shown. Values differed significantly (P = 0.009) between the sexes (β = 0.20; 95% CI, 0.05 to 0.35).
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.653
Erythrocyte GSH concentrations for male (n = 10) and female (14) dogs in both treatment groups over the 28-day study period. Data points represent the raw data, which include repeated measurements for each dog. Mean values (top of gray-shaded bars) and 95% CIs (error bars), derived from a simple linear model of the effect of sex on GSH concentration, are also shown. Values differed significantly (P = 0.009) between the sexes (β = 0.20; 95% CI, 0.05 to 0.35).
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.653
Erythrocyte GSH concentrations for male (n = 10) and female (14) dogs in both treatment groups over the 28-day study period. Data points represent the raw data, which include repeated measurements for each dog. Mean values (top of gray-shaded bars) and 95% CIs (error bars), derived from a simple linear model of the effect of sex on GSH concentration, are also shown. Values differed significantly (P = 0.009) between the sexes (β = 0.20; 95% CI, 0.05 to 0.35).
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.653
No significant effect on erythrocyte GSH concentration was found for RibCys supplementation or study day. The best-supported model was the null model, which received 60% of the model weight (Supplementary Table S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.8.653), representing 3 times the support of the next best-supported model (20% of the model weight). The regression coefficient for the fixed effect of treatment group was –0.01, with a 95% CI (–0.31 to 0.30) that overlapped 0, further illustrating the minimal effect of treatment group on erythrocyte GSH concentration.
Discussion
To the authors' knowledge, the present study was the first to evaluate the safety and efficacy of a RibCys supplement in dogs. Contrary to our hypothesis, no increase in erythrocyte GSH concentration was detected when compared with results for a placebo. Owners of dogs in the RibCys group reported improvements in skin and coat health, osteoarthritis, energy, and appetite. No owners of dogs in the placebo reported any clinical benefits. We found no evidence that erythrocyte GSH concentration differed significantly with age or body weight. We did find that males had slightly higher erythrocyte GSH concentrations than did females, although this difference was not significant. We propose a normal range for all dogs of 1.33 to 2.97mM. The erythrocyte GSH concentrations reported here were similar to a previously reported erythrocyte GSH concentration range for healthy dogs (median, 1.91mM; range, 0.87 to 3.51mM).18 In addition, the healthy dog group in the previous study18 was nearly identical to ours with regard to ownership, age, sex, ratio of mixed-breed to purebred dogs, and body weight, helping to validate our results for erythrocyte GSH concentration.
Oral administration of the RibCys supplement did not increase erythrocyte GSH concentration relative to the placebo in the present study. This finding was unexpected given that a study1 of healthy humans showed increases in baseline blood GSH concentration after 1, 3, and 6 months of RibCys supplementation. In addition, RibCys administration has been shown to increase GSH stores in various tissues, including the liver, kidneys, muscle, urinary bladder, and heart in rodents19 and healthy humans.1 Several possibilities might explain the negative findings for RibCys supplementation in our study. Had we measured tissue GSH concentrations, we might have found greater GSH values in the RibCys-treated dogs than in placebo-treated dogs given that other investigators have documented a lack of correlation between plasma, tissue, and erythrocyte GSH concentrations.20 Alternatively, the use of healthy dogs could have contributed to the lack of increased erythrocyte GSH concentrations because total body GSH concentration may have already been at its peak. The RibCys-treated dogs could have had increased GSH utilization from unrecognized causes of increased oxidative stress, which possibly resulted in stable GSH concentrations despite supplementation—an interpretation supported by the work of others.21 Markers of oxidative stress, including oxidized GSH, were not measured in our study but may help identify underlying oxidative stress and its impact on GSH concentrations in future studies. It must also be considered that cysteine may not be the rate-limiting substrate for GSH production in dogs and that glutamate or glycine may be the limiting substrate.21 The dose and dosing interval chosen may have also contributed to our negative results, but this explanation seems unlikely because the protocol used in this study was based on previously published protocols.1,22 The duration of the present study may have been too brief to detect a significant increase in erythrocyte GSH concentration given that a study1 involving humans shows that the peak change in erythrocyte GSH concentration occurred after approximately 6 months of supplementation. A difference in GSH metabolism between dogs and rodents is supported by the results of a study23 of sick dogs that failed to have an expected increase in erythrocyte GSH concentration when administered antioxidants for 30 days. Given the similarities between our findings and previously reported erythrocyte GSH concentrations in dogs,18 yet another possibility is that RibCys supplementation simply has no effect on erythrocyte GSH concentrations in healthy dogs. A comparison of changes in erythrocyte GSH concentrations between healthy and sick dogs receiving RibCys supplementation would help to further evaluate that possibility.
As we hypothesized, the RibCys supplement was well tolerated in all dogs and the safety profile of RibCys in healthy dogs was consistent with previous reports13,15,16,17,19,22 concerning other species. Dogs in both groups developed mild transient anemia, the cause of which was unclear. That anemia may have been secondary to weekly blood collection, which occurred in both groups. No evidence of hemolysis was observed on the evaluated blood smear preparations, and no reticulocytosis was noted. Because dogs in both treatment groups had mild gastrointestinal signs and transient anemia, we were unable to directly correlate any of those signs to RibCys administration. Although RibCys was well tolerated in healthy dogs, it is unknown whether the same could be expected for dogs with cancer or other underlying diseases. What also remains unclear is how the previously reported differences in erythrocyte GSH concentrations and steady-state free radical concentrations between healthy dogs and dogs with cancer24 may influence the safety and efficacy of RibCys supplementation.
Overall, the number of dogs in the RibCys and placebo groups was small yet the ages and breeds varied, which may have limited our power to observe significant differences between the 2 groups. Because this was a preliminary study, with the ultimate goal of using RibCys in dogs with cancer, the wide range of age and breed was appropriate to mimic this clinical population.
In veterinary medicine, successful cancer treatment is often limited by owners' perception of their pet's quality of life during treatment.25,26,27 The clinical benefits observed in the RibCys-treated dogs of the present study may have been secondary to increased tissue GSH concentrations, which did not translate into increased erythrocyte GSH concentrations. An increase in systemic GSH concentration has been shown to protect against drug-induced hepatotoxicosis14 and cyclophosphamide-induced urotoxicosis17 and to minimize the adverse effects associated with high-dose radiation therapy.16 Other studies6,7,8,9 have shown fewer toxic effects and improvements in quality of life for people receiving chemotherapy, without having a significant impact on treatment outcome. The clinical benefits noted in the present study may have reflected a similar benefit of RibCys supplementation in healthy dogs. Although erythrocyte GSH concentrations did not increase significantly for dogs receiving the RibCys supplement, further investigation of its mechanism of action and clinical benefit in dogs undergoing antineoplastic treatment is warranted given that RibCys was found to be safe and well tolerated, with owner-documented clinical benefits.
Acknowledgments
Funded by Max International LLC. Max International provided the RibCys supplement and placebo but did not contribute to study design or to data collection, analysis, or interpretation.
The authors declare that there were no conflicts of interest. The authors thank Dr. John Richie for his assistance with GSH analysis.
Abbreviations
| GSH | Glutathione |
| Redox | Reduction-oxidation |
| RibCys | d-ribose-l-cysteine |
| ROS | Reactive oxygen species |
Footnotes
Max International, Salt Lake City, Utah.
Idexx Laboratories, Westbrook, Me.
Pathology Core Reference Laboratory, College of Medicine, Penn State University, Hershey, Pa.
R: a language and environment for statistical computing, version 4.0.0, R Foundation for Statistical Computing, Vienna, Austria.
Bates D, Maechler M, Bolker B, et al. lme4: Linear mixed-effects models using ‘Eigen' and S4, version 1.1-14. Available at: CRAN.R-project.org/package=lme4. Accessed May 15, 2020.
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