Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Abstract

Haematological parameters were assessed and all values were observed to fall within the normal range attesting to a general state of good health both before and after dietary supplementation. Comprehensive blood profiles did not show any difference between the first and second phases of the study falling outside of the normal range (Table 2). However, there was a significant increase in the level of serum phosphorous. The reason for this increase is not known. However, a low magnitude change in a single marker wherein the total level still remains in the reference range is not likely to be of clinical significance. In sum, these data suggest the treatment protocol did not lead to any type of overt toxicity.

Table 2: Comprehensive blood profiles.

ALB - albumin; ALP -alkaline phosphatase; ALT - alanine aminotransferase; AMY - amylase; TBIL - total bilirubin; BUN - blood urea nitrogen; CA⁺⁺ - calcium; PHOS - phosphorus; CRE - creatinine; GLU - glucose; NA⁺ - sodium; K⁺ - potassium; TP - total protein; GLOB – globulin. *p < 0.01.

The same was true when comparing baseline and post-treatment blood counts (Table 3). It is notable, however, when comparing these two groups, there was a statistically significant increase in the number of monocytes after the dogs were given the dietary supplement for the treatment period. Although remaining in the reference range, the monocyte number was found to be significantly higher in the second phase of the study (Table 3). This empirical observation is worthy of further investigation.

Table 3: Complete blood count.

GRA - granulocytes; WBC - white blood cells; LYM - lymphocytes; MON - monocytes; RBC - red blood cells; HGB - hemoglobin; HCT - hematocrit; MCV - mean corpuscular volume; MCH - mean corpuscular hemoglobin; MCHC - mean corpuscular hemoglobin concentration; PLT - platelets; MPV - mean platelet volume; *p < 0.0001.

Serum was isolated from whole blood obtained during the two phases of the trial and analysed by LC-MS/MS. The chemopreventive agents used for this are not normally added to dog chow, and selected dosages were well within safety margins based on previous work conducted mainly in human clinical trials. At the end of this first (placebo) phase of the trial, no resveratrol, resveratrol metabolites, curcumin, curcumin metabolites, or ellagic acid were detected in serum. Genistein-4′-glucuronide (327.7 ± 211.13 ng/mL) and genistein sulfate (961 ± 680.1 ng/mL) were detected in serum of six dogs. It is likely the genistein metabolites were derived from the dog chow, which can contain soybean products. Quercetin levels were below detectable levels in five dogs, but three dogs had quercetin concentrations of 2 or 3 ng/mL. Quercetin occurs in many botanicals and was also probably a constituent of the dog chow (Table 4). After a two-month rest (wash-out) period, the dogs were started on the second phase of the trial by administering two biscuits per day containing the chemopreventive agents. The second phase lasted 3 weeks. Resveratrol and its metabolites were detected in serum of all dogs but one during the second phase. The most abundant form of resveratrol in dog serum was resveratrol-3-sulfate. Much higher concentrations of genistein metabolites were observed during the second phase (genistein-4′-glucuronide, 7083.74 ± 128.18 ng/ml; genistein-7-glucuronide, 861.74 ± 329.61 ng/ml), with the sulfate being dominant (genistein sulfate, 26958.61 ± 2300 ng/ml) (Table 4). A diet rich in genistein has been associated with lower rates of prostate and breast cancer²⁸. When administered orally, genistein is rapidly absorbed and metabolized in humans and animals as are other isoflavones²⁹. In the current study, no unconjugated genistein was detected after either the first or the second phases (Table 4). No ellagic acid or curcumin was detected in serum, although curcumin glucuronide was detected at low levels in serum of two dogs after the second phase.

Table 4: Concentrations of chemopreventive compounds and its metabolites in dog serum.

Starting with whole blood from the canine subjects, peripheral blood lymphocytes were isolated. Two measures of DNA damage were evaluated: endogenous DNA damage (DNA damage without H₂O₂ exposure) and H₂O₂-inducible DNA damage. We did not find any statistically significant difference in endogenous lymphocyte DNA damage between the two phases of the study (Fig. 1). In dog lymphocytes obtained during the first phase of the trial, H₂O₂-induced DNA strand breakage increased from 54 ± 15 to 310 ± 30 units. Remarkably, in second phase of the trial, following administration of the dietary supplement, lymphocyte DNA damage induced by treatment with 100 μM H₂O₂ was significantly decreased from 310 ± 30 to 180 ± 16 units (Fig. 1, grey bars). For further confirmation of these data, cell preparations were treated with endonuclease III and the measurements were repeated. Levels of endogenous oxidized pyrimidines did not differ during the first or second phases of the trial. However, H₂O₂ treatment increased the level of oxidized pyrimidines, and consumption of the dietary supplement protected lymphocytes against pyrimidine oxidation (Fig. 1, black bars).

Figure 1: Chemopreventive supplements inhibit H₂O₂-induced DNA strand breakage in dog lymphocytes.

Lymphocytes were washed, suspended in 1% (w/v) LMP agarose and pipetted onto frosted glass microscope slides. For H₂O₂ treatment, 10 μl of 1 mM H₂O₂ were added to 90 μl of cells in PBS (100 μM final concentration); after 5 min on ice, the cells were collected by centrifugation and applied to slides. Slides were washed three-times with buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8) and incubated for 45 min at 37 °C with either 50 μl buffer or endonuclease III in buffer (1 μg protein/ml). Lymphocytes were stained with SYBR Gold and image analysis was performed using a Leica confocal microscope (Fig. 2). Gray bars are without endonuclease treatment, black bars are with treatment. **p < 0.01, t-test (n = 16).

To further explore the ramifications of chemopreventive agent administration, total RNA was extracted from lymphocytes, cDNA was generated, and evaluated using a custom made PCR array relevant to dog oxidative stress (Table 5). Of the 84 genes evaluated, the expression of 11 was found to be significantly altered following administration of the dietary supplement (Table 6). The expression of five genes, including CYBB, DUSP, GSR, UCP2 and VIMP, were significantly increased, whereas those of six genes, including ATOX1, CCL5, EPX, MPV17, PRNP and SOD3, were down-regulated (Fig. 2). Among these, VIMP and MPV17 were most highly up- and down-regulated, respectively.

Figure 2: Relative expression levels of oxidative stress-related genes in dog lymphocytes.

Lymphocytes were isolated from the same dogs after the two phases of the study. Total RNA was extracted and reverse-transcribed to cDNA. The resultant cDNA template was mixed with RT2 SYBR Green qPCR Mastermix and distributed equally as 25 µl aliquots in each well of the customized array plate. After the initial incubation at 95°C for 10 min to activate HotStart Taq DNA polymerase, real-time quantitative PCR was performed with the thermal cycling conditions as 40 cycles at 95°C for 15 sec followed by 60°C for 1 min. The fluorescence signal from SYBR Green intercalating into double-stranded DNA was recorded at the end of each elongation phase. The fold changes of 84 genes between the two phases of the study were analyzed using the web-based software provided by Qiagen (Data Analysis Center). The heat map represents fold changes in gene expression between control (the first phase) and dietary intervention (the second phase) groups. Red, green, or black squares in the heat map denote up-regulated, down-regulated, or unchanged, respectively, compared to the control group.

Table 5: Oxidative stress related genes custom made for dogs.

Gene Bank             Symbol        Description

____________________________________________________________________________________________________________________

NM_001003026          ALB            Albumin
 

Table 6: Transcriptional regulation of oxidative stress-related genes in dog lymphocytes.Positive values indicate up-regulation; negative values, down-regulation.

Discussion

In situations wherein a tumor is diagnosed and completely excised prior to invasion or metastatic spread, treatment should be definitive. In canine cases, this may involve amputation or radical surgery. However, the situation is even worse when presented with malignant metastatic disease wherein life expectancy is reduced and treatment options are much more limited. Consequently, avoiding all such situations, ostensibly by means of cancer chemoprevention, remains a compelling approach. Despite issues associated with the development of cancer chemopreventive agents, proof-of-principal has been established with drugs such as tamoxifen and finasteride³⁰. In nearly every case, for the discovery or development of new cancer chemopreventive agents, laboratory animals (primarily rodents) have been used as models. In more advanced stages of development, additional animals, including canines, are used to establish pharmacokinetics (absorption, distribution, metabolism, and excretion), dosage forms, toxicity, etc. Nonetheless, the ostensible goal of such work is to provide agents for the prevention of cancer with human beings.

Similar to humans, dogs manifest a broad range of cancers such as melanoma, non-Hodgkin lymphoma, osteosarcoma, soft tissue sarcoma, and prostate, mammary, lung and colorectal carcinomas. Approximately 1 in 3 dogs will be diagnosed with cancer during their lifetime, and cancer currently accounts for about half of the deaths of all dogs older than 10 years^2,31. Of course from a humanitarian point-of-view prevention of canine cancer is gratifying, but owners of pets and service animals would also benefit. Thus, in this study, rather than using the canine as a model for human beings, our goal was to focus on potential benefits for the animal.

This is the first report to document oral administration of this schedule of combined chemopreventive agents in a canine study. The chemopreventive formulation devised for this study is somewhat arbitrary but based on a variety of pragmatic and theoretical considerations. First of all, it was of interest to select agents that (1) are natural products, (2) ostensibly function by pleotropic mechanisms (e.g., inhibit various stages of carcinogenesis such as initiation, promotion, and progression, either singly or in combination), (3) have well-known safety profiles or generally recognized as safe (GRAS) status, (4) have been extensively reported in the scientific literature as chemopreventive agents, and (5) are undergoing or have undergone evaluation in human clinical trials. The following agents were judged as having met these criteria: Reveratrol (‘typical’ representative dose, 1 g/d/person), lycopene (15 mg/d), α-tocopherol (50 mg/d), l-selenomethionine (0.2 mg/d), ellagic acid (250 mg/d), indole-3-carbinol (400 mg/d), sulforaphane (50 mg/d), quecetin (1 g/d), allicin (3 mg/d), genistein (500 mg/d), daidzein (300 mg/d), curcumin (1 g/d), and EGCG (300 mg/d). At this point in the selection process, some agents were eliminated since adequate quantities may be found in the average diet (e.g., vitamin D, selenium, and α-tocopherol), high expense or issues with general availability (e.g., sulforaphane), poor organoleptic properties (e.g., allicin), or innate difficulty in the biscuit production process.

In the end, a combination including resveratrol, genistein, ellagic acid, curcumin and quercetin was selected for administration at the dosage levels summarized in Table 1. The rationale of the dosage selection is described in Methods (Dog biscuit production). Admittedly, additional or alternative agents could have been included in this trial. Nonetheless, in addition to the rationalization presented above, another advantage was that each of the test agents is known to function by pleiotropic mechanisms of action that are relevant to disease prevention. For example, resveratrol has been reported to exert antioxidant, anti-inflammatory, anti-infective, cardioprotective, neuroprotective, antiobesity, and chemopreventive activities^32,33,34,35. It has not been evaluated for cancer chemoprevention in dogs, but analytical work for the determination of trans-resveratrol and derivatives in dog plasma has been described^21,22, as well as preclinical toxicity studies²³. Ellagic acid occurs in many foods and has antioxidant, antibacterial, antiviral and cancer-preventing properties³⁶. It exerts anticancer effects in various cancer cell lines and animal tumor models³⁷. Curcumin, a natural polyphenolic antioxidant can mediate a large number of biological responses³⁸. Some studies have reported pharmacokinetics in dogs, and stabilizing curcumin with phosphoric acid allows accurate quantitative determinations of curcuminoids in dog plasma³⁹. The phytoestrogen genistein is found in soy-based products. It has been suggested that genistein can prevent both prostate and breast cancer²⁸, particularly in Southeast Asia populations where soy products are consumed at high levels⁴⁰. A LC-MS-MS method has been developed for the determination of genistein in dog plasma following oral administration¹⁸. Finally, quercetin mediates diverse anti-cancer effects⁴¹. It is a pleiotropic molecule with limited toxicity on normal cells. Simultaneous targeting of multiple pathways may help to eliminate malignant cells and retard the onset of drug resistance⁴².

The development and production of a biscuit-type dosage form for relatively large canines presented some challenges since it was important to preserve the integrity of the chemopreventive agents. Thus, the common practice of baking the final product was avoided. During the development phase, many formulations were designed to form durable biscuit-type tablets and changes were made in an effort to reduce bellyband cracks and improve compressibility, compatibility, and flavour. The compaction of each active ingredient was individually tested, which revealed that the compounds themselves aid the compression of the tablets and helped balance the unfavourable compaction characteristics of the other biscuit ingredients.

Another issue that will ultimately be faced in developing a canine chemopreventive product is expense. Based on the quantities and price schedule for the chemopreventive agents purchased and used in this study, the expenditure was $1.65/biscuit. It is difficult to know an amount consumers would find to be acceptable for such as product, but given the high expense associated with treatment (vide supra) and the gratification accompanying disease prevention, an expenditure of this magnitude seems reasonable. Another aspect related to expense is when the dog would begin biscuit consumption. While it is not possible to provide an answer based on existing evidence, since it has been noted that more than half of dogs over 10 years of age are likely to develop cancer⁴³, it would seem that early administration would be beneficial. One suggestion would be to start consumption at the time of full growth, which would be roughly 1 year for smaller breeds, and 2 years for larger breeds.

Using the formulation devised for this study, we were able to demonstrate detectable serum levels of resveratrol, genistein metabolites, and quercetin (and metabolites), indicating they cross canine enterocytes and enter blood circulation. This promoted a protective effect on oxidative DNA damage. Since lymphocytes are readily assessable and play important roles in both the physiological and pathological processes, it is reasonable to regard them as a useful indicator for oxidative DNA damage⁴⁴, in humans⁴⁵ as well as canines⁴⁶ and other species¹⁷. Our general hypothesis is that lymphocytes serve as a surrogate for other cell types, and protection from DNA damage is consistent with cancer chemoprevention.

The mechanism of protection remains to be defined, but to provide some indication of the effect of the chemopreventive formulation on oxidative stress gene expression, array analysis was performed. Of the up-regulated genes, three belong to oxidative stress responsive group (DUSP1, GSR, VIMP), CYBB is an antioxidant, and UCP2 is related to superoxide metabolism. Many selenoproteins (VIMP) participate in intracellular redox homeostasis and play antioxidant roles⁴⁷. The expression of selenoprotein S (SelS) is related to inflammation and insulin resistance suggesting that SelS may provide a link between inflammation and oxidative stress pathways through its role as an antioxidant⁴⁸. Down-regulated genes are represented by two genes (ATOX1 and MPV17) associated with ROS metabolism, two oxidative stress responsive genes (CCL5 and PRNP), and two others from the antioxidant group (SOD3 and EPX)^{31,49,50,51,52} (Table 6). Overall, in the context of gene expression, up-regulation (e.g., GSR and UCP2) and down-regulation (e.g., SOD3 and the transcription factor for SOD3, Atox1)⁵³ may improve antioxidant response or decrease inflammation.

In order to assess the general health of the animals, comprehensive blood profiles were examined during the first and second phases of the study. No changes of veterinary concern were observed, but the population of monocytes and the concentration of phosphorous were found to be increased after the dogs were given the dietary supplement for the treatment period. Nonetheless, every parameter determined remained in the reference range.

In sum, the results reported herein show significant promise and support the notion of perfecting a nontoxic dietary supplement that can be of value in enhancing the health and well-being of “man’s best friend” the canine. Of course this would be a meaningful accomplishment for the dog him- or herself, but owners of domestic pets and service animals would also derive great benefit. Further, it is encouraging that regulatory requirements would be less stringent than those mandated for human consumption, and ultimately, the work with canines may provide some insight relevant to human disease prevention.

Methods

Comet assay

The extent of DNA damage in peripheral blood lymphocytes was measured by single-cell gel electrophoresis (the alkaline comet assay)^17,56,57. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. DNA was allowed to unwind under alkaline conditions. Breaks in the DNA molecule disrupt its complex supercoiling allowing free DNA loops to migrate towards the anode during electrophoresis. DNA damage to the cells can be thus visualized as “comets” (Fig. 3). Two measures of DNA damage in lymphocytes were evaluated: endogenous DNA damage (DNA damage without ex vivo H₂O₂ exposure) and H₂O₂-inducible DNA damage. Analysis was performed according to the method of Singh et al.⁵⁷ and Tice et al.⁵⁸ with some modifications⁵⁹. A Trevigen Comet assay kit (Gaithersburg, MD) was used for single cell gel electrophoresis. Cells were immobilized in a bed of low melting agarose on a Trevigen CometSlide. A cell suspension containing 1 × 10⁵ cells per ml was combined with 500 μl of LMAgarose providing agarose in a ratio 1:10 for optimal results when spread at 50 μl of mixture per slide well. Slides were covered with a glass cover-slip and left at 4 °C in the dark for 10 min. Increasing gelling time to 30 min improved adherence of the samples. Slides were placed for a minimum of one hour in lysis solution (the buffer formulation is proprietary) to remove membranes, cytoplasm and nuclear proteins. The CometSlide was then immersed in alkaline unwinding solution for 20 min at room temperature or 1 h at 4 °C in the dark. Horizontal electrophoresis was conducted at 21 volts for 30 min at 4 °C. After neutralizing, the gels were soaked in 70% ethanol for 5 min and dried at 37 °C for 10–15 min. Gels were treated with 100 μl SYBR Gold nucleic acid stain (Life Technologies) and viewed by fluorescent microscopy using a Leica TCS SPE confocal microscope (Leica, UK) at Abs/Em 496/540 nm. Slides were examined at 400× magnification using image analysis software (CometScore from TriTek Corp., Sumerduck, VA).

Figure 3: Comet images of lymphocyte DNA damage from control dogs and dogs receiving dietary supplementation.

Frozen lymphocytes were thawed, centrifuged, resuspended in PBS and incubated 5 min on ice with 100 μM H₂O₂. DNA damage was measured with the comet assay. Type 0, no DNA damage detected in lymphocytes; type 1 and 2 presented at the most in lymphocytes with endogenous DNA damage; type 3 and 4 are in H₂O₂ or endonuclease III inducible DNA damage.

From each replicate slide, 50 nuclei were scored and the percentage of tail DNA intensity was used to evaluate the extent of DNA migration and damage⁴⁶. Fluorescently stained nucleoids from each gel were assessed and classified according to the relative intensity of fluorescence in the tail undamaged (Fig. 3). Each cell was visually scored according to the following criteria: no damage (type 0), mild to moderate damage (type 1 and 2), and extensive DNA damage (type 3 and 4). Under the assay conditions used in this experiment, the intensity of comet tails reflects electrophoretic migration of DNA resulting from strand breaks. This parameter was used as arbitrary units. Tail length tends to increase rapidly with dose at low levels of damage, but soon reaches its maximum. We did not use tail moment units since it combines the information of tail length and tail intensity, but suffers from lack of linearity⁴⁶. To detect specifically oxidized pyrimidines in DNA, recombinant bacterial DNA repair enzyme, endonuclease III from E. coli, was used⁴⁵. This provides a specific and sensitive measure of oxidative DNA damage⁴⁵. The slides were washed three-times after lysis for 5 min at 4 °C with buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8). After blotting dry with tissue paper, the gels were incubated for 45 min at 37 °C with either 50 μl buffer or endonuclease III in buffer (1 μg protein/mL). All samples were analysed in duplicate. Levels of endogenous oxidized pyrimidines demonstrated a similar pattern as endogenous DNA damage, i.e., no significant difference between first and second phases (Fig. 1).

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