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Vitamin D3 suppresses the early stages of chemically induced hepatocarcinogenesis in rats: a dose-response analysis

  • Mariana B. Tablas1Email author,
  • Renata L. Goto1,
  • Brunno F. R. Caetano1,
  • Sérgio A. A. dos Santos1 and
  • Luis F. Barbisan1
Nutrire201843:12

https://doi.org/10.1186/s41110-018-0065-2

Received: 15 September 2017

Accepted: 2 February 2018

Published: 23 April 2018

Abstract

Background

The aim of this study was to investigate dose-response effects of vitamin D3 (VD3) supplementation on the early stages of diethylnitrosamine (DEN) and carbon tetrachloride (CCl4)-induced hepatocarcinogenesis in rats.

Methods

The animals were randomly allocated into six experimental groups (10 rats each) treated as follows: group 1: no treatment; groups 2–6: single intraperitoneal injection of N-diethylnitrosamine; groups 2–6: intragastric CCl4; groups 3–6: intragastric VD3 at 10,000, 20,000, 40,000, and 60,000 IU/kg b.w., respectively.

Results

Serum 25-hydroxyvitamin D (25-OHD) levels in the VD3-supplemented groups were significantly higher than those in the control groups (G1 and G2, p < 0.001). Serum levels of phosphate were higher in the groups supplemented with VD3 at 10,000 and 60,000 IU/kg (G3 and G6, p < 0.005). VD3 higher doses reduced cell proliferation and the number of larger placental glutathione S-transferase (GST-P)-positive hepatocellular preneoplastic lesions. Neither the DEN/CCl4 regimen nor the VD3 supplementation altered vitamin D receptor (VDR) protein expression in the liver.

Conclusion

The results indicate that high-dose VD3 supplementation reduced the development of DEN/CCl4-induced preneoplastic lesions in the liver.

Keywords

Vitamin DHepatocarcinogenesisPreneoplastic lesionsCell proliferation

Background

Vitamin D (VD) deficiency is a trending global health issue [13]. According to the World Health Organization (WHO), over a billion people worldwide are VD deficient or insufficient [4, 5]. This highly prevalent condition has been associated with an increased risk of developing chronic diseases such as diabetes, obesity, and cancer [68]. Over the past few years, a growing number of epidemiological studies have reported that VD deficiency is very common in patients with chronic liver diseases [912]. Clinical evidence highlights the prevalence of VD deficiency among patients with chronic hepatitis C, cirrhosis, and hepatocellular carcinoma (HCC) [1316]. Therefore, VD supplementation has become an appealing treatment in order to prevent, suppress, or ameliorate a number of chronic liver diseases [7, 1720].

Dietary VD can be obtained through naturally food sources containing VD2 (ergocalciferol), which is found in plant sources, or VD3 (choleocalciferol), found in animal sources [21, 22]. However, the major source of VD3 comes from natural synthesis in the human skin through exposure to natural sunlight [23, 24]. The energy of ultraviolet B sun rays stimulates vitamin D synthesis in the skin from the conversion of 7-dehydrocholesterol to the secosteroid VD3. VD3 is further hydroxylated in the liver to a circulating pro-hormone 25-hydroxyvitamin D (25OHD3, calcidiol). This hydroxylation, which occurs exclusively in hepatocytes, is mediated by CYP27A1 and CYP2R1 that show different specificity and affinity for VD3 [25]. The conversion of 25OHD3 to its final active form 1,25-dihydroxyvitamin D3 (1,25OHD3, calcitriol) is subsequently achieved in the kidneys through enzymatic activity catalyzed by the mitochondrial cytochrome 1α-hydroxylase (CYP27B1) enzyme [1, 26].

The biological functions of the active form of VD3 are mediated by the nuclear vitamin D receptor (VDR), a high-affinity phosphoprotein receptor that binds to the 1,25OHD3 hormone and regulates gene expression in a number of cellular processes, including cell proliferation, differentiation, apoptosis, and immunomodulation [20, 27]. Therefore, the hepatic expression of VDR is suggested to be inversely associated with the severity of liver damage [2528]. Low liver VDR expression has been implicated in the development of non-alcoholic steatohepatitis (NASH), fibrosis, and HCC [27].

To date, only a few rodent studies have proposed that VD3 supplementation reduces chemically induced rat hepatocarcinogenesis or liver fibrosis, with a positive outcome observed in the late stages of these diseases [17, 29, 30]. The purpose of the present study is therefore to investigate dose-response effects of VD supplementation in the early stages of diethylnitrosamine (DEN) and carbon tetrachloride (CCl4)-induced hepatocarcinogenesis in rats.

Methods

Animals

Four-week-old male Wistar rats, acquired from the São Paulo University Medical School (Ribeirão Preto, SP, Brazil), were housed in polypropylene cages under controlled temperature (22 ± 2 °C), humidity (55 ± 10%), and lighting (12 h light/12 h dark cycle) with free access to water and commercial chow (Presence®, Paraná, Brazil).

Study design

The animals were randomly assigned into six experimental groups of 10 rats each. Group 1: untreated group (sham); groups 2–6: received a single intraperitoneal injection of 200 mg/kg body weight (b.w.) of N-diethylnitrosamine (DEN, Sigma-Aldrich Co., St. Louis, CA, USA) as an initiating agent. Groups 2–6 received intragastric administrations of 1.0 ml/kg b.w. of carbon tetrachloride (CCl4, Dinâmica®, SP, Brazil), once a week, as a promoting agent for 10 weeks and stopped [31, 32]. Groups 3–6 received intragastric administration of VD3 (Dry Vitamin D3 100, BASF—Ludwigshafen, Germany) at 10,000, 20,000, 40,000 and 60,000 IU/kg b.w., respectively, on alternate days for 16 weeks. Both CCl4 regimen and VD3 supplementation started 2 weeks after DEN administration.

At the end of the experiment, the animals were euthanized by exsanguination under sodium pentobarbital anesthesia (30 mg/kg b.w.). Peripheral blood samples were collected for measuring serum aspartate amino transferase (AST, automated kinetic method, Cobas C501—Roche, USA), cholecalciferol (25OHD3, high-performance liquid chromatography—HPLC), calcium, and phosphate (Bioclin, Germany). After the sacrifice, the liver was removed and weighed. Liver tissue fragments were collected and either stored at − 80 °C for protein extraction or fixed in 10% phosphate-buffered formalin for histological and immunohistochemical analyses (Fig. 1).
Figure 1
Fig. 1

Study design

Immunohistochemistry

Immunoreactivity for Ki-67 and placental glutathione S-transferase (GST-P) was detected using a universal labeled Streptavidin-Biotin system (LSAB System-HRP, DakoCytomation, Denmark). Briefly, deparaffinated 5-μm liver sections on silanized slides were sequentially treated with citrate buffer (120 °C, 5 min) in a Pascal Pressure Chamber (DakoCytomation, Denmark), 3% H2O2 in phosphate-buffered saline (PBS) (10 min), skim milk (60 min), rabbit monoclonal anti-Ki-67 (1:100 dilution, Abcam, UK) or GST-P (1:1000 dilution, Medical & Biological Laboratories, Japan) antibodies overnight (4 °C) and biotinylated universal link and streptavidin HPR (20 min each). Color development was achieved using 3,3-diaminobenzidine (DAB, Sigma-Aldrich, USA) and counterstained with Harris’s hematoxylin. The proliferative index was defined as the number of Ki-67-positive hepatocytes per microscope field (40 microscopic fields per animal at × 40 objective). Preneoplastic liver lesions (PNL) were assessed by immunohistochemical staining for GST-P, a biomarker for detection of preneoplastic and neoplastic lesions [33, 34]. GST-P-positive PNL were measured using a KS-300 image analysis software (Kontron Elektronic, Germany). Data were expressed as number of GST-P-positive PNL per liver area (cm2), classified into three different sizes: < 0.5 mm2, 0.5–1.0 mm2, and > 1.0 mm [34, 35]. Larger GST-P-positive lesions are indicative of promoting effects and higher growth rates [34].

Western blot analysis

Liver samples were homogenized in lysis buffer (1% Triton X-100 and 2 μl/100 ml protease inhibitor, Sigma-Aldrich, USA). After this procedure, the extracted material was centrifuged (4000 rpm, 4 °C, 20 min) and the supernatant collected for protein quantification by Bradford’s method. Aliquots of liver homogenates containing 70 μg of total protein were heated (95 °C, 5 min) in sample-loading buffer and, then, electrophoretically separated in a 12% SDS–PAGE gel under reducing conditions and transferred to nitrocellulose membranes (Sigma-Aldrich, USA). Membranes were blocked with skim milk in TBS-T (0.05 M Tris, 0.15 M NaCl, pH 7.2, 1% Tween-20) for 1 h. The nitrocellulose membranes were subsequently incubated with polyclonal antibodies rabbit anti-VDR (1:1000 dilution, Santa Cruz Biotechnology, CA, USA) and anti-CYP27A1 (1:1000 dilution, Santa Cruz Biotechnology, CA, USA) or goat polyclonal anti-actin (1:1000 dilution, Santa Cruz Biotechnology, USA) primary antibodies in 5% BSA solution overnight. After five wash steps with PBS-T, membranes were incubated with specific horseradish-conjugated secondary antibodies, according to the primary antibodies used, for 2 h at room temperature. Finally, after five wash steps, the membranes were submitted to immunoreactive protein signals (GE Healthcare Life Sciences, UK). Signals were captured by a G:BOXChemi system (Syngene, UK) controlled by an automatic software (GeneSys, Syngene, UK). Band intensities were quantified using densitometry analysis software (Image J software, Austria). Finally, VDR and CYP27A1 protein expression was reported as fold change according to actin protein expression, used as a normalizer.

Statistical analysis

Body weight, liver weight, food intake, as well as cell proliferation index, the number of GST-P-positive lesions, and calcium and phosphorus levels were analyzed by the ANOVA test and post hoc Tukey’s test. Statistical analysis was performed using the Jandel Sigma Stat Software (Jandel Corporation, San Rafael, CA, USA). Graphics were generated by the GraphPad Prism software (Version 6.01, La Jolla, CA). Statistical differences were considered significant when p < 0.05.

Results

Body weight, food intake, and liver weight

A significant reduction in body weight gain and final body weight was observed in the groups receiving VD3 supplementation at doses of 40,000 and 60,000 IU/kg b.w. (G5 and G6, p < 0.001) when compared to the untreated and DEN/CCl4-treated groups (G1 and G2, respectively). The weight loss in the VD3 high-dose groups was accompanied by a significant decrease in food intake (G5 and G6, p < 0.001) (Table 1). The relative liver weight was significantly lower in the groups supplemented with high doses of VD3 (G5 and G6, p < 0.001) than in the untreated and DEN/CCl4-treated groups (G1 and G2, respectively).
Table 1

Effects of vitamin D3 supplementation on final body weight, body weight gain, food intake, and liver relative weight among the experimental groups

Groups/treatments (kg b.w.)

No. of rats

Final body weight (g)

Body weight gain (g)

Food intake (g/rat/day)

Liver relative weight (%)

Non-supplemented

 G1

Control (SHAM)

10

558 ± 46.1

238 ± 34.0

2.30 ± 0.08

29.9 ± 2.63

 G2

DEN + CCl4

10

498 ± 45.6

195 ± 37.0

2.73 ± 0.34

29.0 ± 3.21

Supplemented

 G3

VD3 10,000 IU

10

508 ± 77.2

205 ± 57.2

2.71 ± 0.15

30.1 ± 3.30

 G4

VD3 20,000 IU

10

515 ± 55.5

206 ± 40.8

2.70 ± 0.17

29.7 ± 3.02

 G5

VD3 40,000 IU

10

452 ± 69.8*

149 ± 52.8*

2.89 ± 0.27*

27.3 ± 4.78*,**

 G6

VD3 60,000 IU

10

407 ± 73.4*,**

106 ± 51.7*,**

2.88 ± 0.18*

24.8 ± 4.82*,**

Values are means ± SD (standard deviation). Means were compared by one-way ANOVA followed by Tukey’s multiple comparisons test. DEN = N-diethylnitrosamine (200 mg/kg, i.p. single dose); CCl4 = carbon tetrachloride, i.g. 1.0 ml/kg, once a week for 10 weeks. G1 = untreated group (SHAM); G2 = DEN + CCl4; G3 to G6 = DEN + CCl4 + VD3 (choleocalciferol) at 10,000 IU, 20,000 IU; 40,000 IU and 60,000 IU/kg (i.g., on alternate days for 16 weeks), respectively. Liver relative weight (%) = absolute weight (g)/final body weight (g) × 100

*, **Significantly different from G1 or G2, respectively, p < 0.001

Serum levels of 25OHD3, AST, calcium, and phosphate

Serum 25-hydroxyvitamin D (25-OHD) levels in VD3-supplemented groups were significantly higher than those in the control groups (G1 and G2, p < 0.001) (Fig. 2).
Figure 2
Fig. 2

Serum levels of 25 (OH) D. Values are means ± SD compared by one-way ANOVA followed by Tukey’s multiple comparisons test. One asterisk indicates significantly different from G1, p < 0.0001. Two asterisks indicate significantly different from G2, p < 0.0001, and three asterisks indicate significantly different from G3, p < 0.0001. G1 = untreated group; G2 = DEN + CCl4; G3 = DEN + CCl4 + VD3 10,000 IU; G4 = DEN + CCl4 + VD3 20,000 IU; G5 = DEN + CCl4 + VD3 40,000 IU; G6 = DEN + CCl4 + VD3 60,000 IU

Furthermore, serum levels of phosphate were higher in the groups supplemented with VD3 at 10,000 and 60,000 IU/kg (G3 and G6, p < 0.005) than in remaining groups. However, serum calcium and AST levels did not differ among groups (Table 2).
Table 2

Serum alanine aminoaspartate (AST), calcium, and phosphorus levels in the different experimental groups

Groups/treatments (kg b.w.)

No. of rats

AST (U/l)

Serum calcium (mg/dl)

Serum phosphorous (mg/dl)

Non-supplemented

 G1

Control (untreated)

10

45.3 ± 7.37

6.77 ± 0.49

7.82 ± 0.09

 G2

DEN + CCl4

10

53.1 ± 12.8

8.18 ± 0.66

8.24 ± 0.35

Supplemented

 G3

VD3 10,000 IU

10

58.7 ± 11.2

9.22 ± 1.42

8.88 ± 0.66*

 G4

VD3 20,000 IU

10

54.7 ± 10.2

8.63 ± 1.56

8.21 ± 0.61

 G5

VD3 40,000 IU

10

58.9 ± 13.0

7.93 ± 0.97

8.27 ± 0.44

 G6

VD3 60,000 IU

10

58.6 ± 20.9

9.24 ± 1.47

8.33 ± 1.02*

Values are means ± SD (standard deviation). Means were compared by one-way ANOVA followed by Tukey’s multiple comparisons test. DEN = N-diethylnitrosamine (200 mg/kg, i.p. single dose); CCl4 = carbon tetrachloride, i.g. 1.0 ml/kg, once a week for 10 weeks. G1 = untreated group (SHAM); G2 = DEN + CCl4; G3 to G6 = DEN + CCl4 + VD3 (choleocalciferol) at 10,000 IU, 20,000 IU; 40,000 IU and 60,000 IU/kg (i.g., on alternate days for 16 weeks), respectively

*Significantly different from G1, p < 0.005

Cell proliferation and preneoplastic lesion development

The average for Ki-67 labeling index (Ki-67 LI%) in the DEN/CCl4-induced group (G2) was significantly higher than that in the untreated group (G1, p < 0.001). There was a reduction in cell proliferation indexes in the group that received the higher doses of VD (60,000 IU/kg (p = 0.0002, Fig. 3) when compared to the DEN/CCl4-induced group (G2).
Figure 3
Fig. 3

Immunohistochemical detection of Ki-67-positive hepatocytes. a Cell proliferation index in the different groups. b Representative image with hepatocytes showing nuclear positivity for Ki-67(final magnification × 400). Values are means ± SD, compared by one-way ANOVA followed by Tukey’s multiple comparisons test. One asterisk indicates significantly different from G1, p < 0.001. Two asterisks indicate significantly different from G2, p < 0.0002. G1 = untreated group; G2 = DEN + CCl4; G3 = DEN + CCl4 + VD3 10,000 IU; G4 = DEN + CCl4 + VD3 20,000 IU; G5 = DEN + CCl4 + VD3 40,000 IU; G6 = DEN + CCl4 + VD3 60,000 IU

With regard to the number of GST-P-positive PNL per liver area, VD3 supplementation significantly reduced the number of GST-P-positive PNL larger than 1.0 mm2 when compared to the positive control group (G2, p < 0.0001) (Fig. 4).
Figure 4
Fig. 4

ac Evaluation of number of GST-P-positive PNL per liver area (mm2). d Representative image of a GST-P-positive hepatic foci (final magnification × 400). 1Values are means ± SD, compared by one-way ANOVA followed by Tukey’s multiple comparisons test. One asterisk indicates significantly different from G3, G4, G5, and G6, p < 0.0001. G1 = untreated group; G2 = DEN + CCl4; G3 = DEN + CCl4 + VD3 10,000 IU; G4 = DEN + CCl4 + VD3 20,000 IU; G5 = DEN + CCl4 + VD3 40,000 IU; G6 = DEN + CCl4 + VD3 60,000 IU

VDR and CYP27A1 protein expression

Hepatic VDR protein expression was similar in all groups, independently of the DEN/CCl4 regimen or VD3 supplementation. In contrast, CYP27A1 protein expression was significantly higher in the liver from DEN/CCl4-treated group (G2) than in the remaining groups (G1, G4, G5, and G6, p = 0.007) (Fig. 5).
Figure 5
Fig. 5

a Western blotting for the hepatic CYP27A1 and b VDR proteins. Values are means ± SD. Means were compared by one-way ANOVA followed by Tukey’s multiple comparisons test. One asterisk indicates significantly different from G1, p = 0.0067. Two asterisks indicate significantly different from G2, p = 0.0067. G1 = SHAM; G2 = DEN + CCl4; G3 = DEN + CCl4 + VD3 10,000 IU; G4 = DEN + CCl4 + VD3 20,000 IU; G5 = DEN + CCl4 + VD3 40,000 IU; G6 = DEN + CCl4 + VD3 60,000 IU

Discussion

The aim of this study was to investigate dose-response effects of VD3 supplementation on the early stages of DEN/CCl4-induced hepatocarcinogenesis in rats. Our results indicate that VD3 supplementation at 40,000 and 60,000 IU/kg significantly decreased body weight gain, accompanied by a reduction in food intake. These findings are in agreement with the literature indicating that VD3 intake may contribute to weight loss [36]. Experimental studies and intervention trials have proposed that VD3-mediated weight loss may be attributed to the modulation of fat oxidation profiles, thus increasing the overall metabolism [36, 37]. VD3 has also been shown to be capable of modulating insulin sensitivity and thereby decrease hunger, improve satiety, and reduce food intake [38, 39]. Furthermore, relative liver weight was also decreased in the groups supplemented with high doses of VD3, but without no specific hepatocellular alterations or ALT levels changes. Although VD3 toxicity is low, the doses used in this study were lower than 100,000 UI because higher doses can cause vitamin D intoxication, hypercalcemia, hyperphosphatemia, and ultimately death [40, 41]. However, the possibility of long-term high-dose toxicity should be investigated.

The steroid hormone 1,25OHD3 (calcitriol) plays a crucial role in calcium and phosphorus homeostasis. The parathyroid hormone regulates conversion of 25OHD3 to its active metabolite, 1,25OHD3, which increases calcium and phosphorus levels in blood by increasing intestinal absorption [42, 43]. Serum calcium is highly regulated, promptly mobilized, and stored in the bones, maintaining serum calcium concentrations within a normal physiological range. Dietary phosphorus excess is excreted by the kidneys under the regulatory activity of the fibroblast growth factor 23 protein (FGF23), decreasing CYP27B1 expression and VD3 activation and promoting phosphorus excretion in urine [43, 44]. Although no changes in serum calcium levels were found in any of the study groups, higher levels of serum phosphate were observed in those supplemented with VD3 at 10,000 and 60,000 IU (G3 and G6, respectively) than in the other groups. These results support the hypothesis that VD3 supplementation was effective and promoted phosphorus mobilization via VD3 metabolic activation [45, 46]. However, since high dietary inorganic phosphate can initially promote but later inhibit lung cancer progression in mice [47], the increase in serum phosphate should be investigated in long-term VD3 interventions.

VD3 has been demonstrated to suppress cellular proliferation in highly cell-specific manners, via apoptosis, cell cycle progression, and differentiation [48, 49]. In our study, DEN/CCl4 regimen (G2) increased hepatic cell proliferation when compared to the untreated group (G1). However, VD3 supplementation at higher doses (40,000 and 60,000 IU/kg) suppressed cell proliferation in comparison to the DEN/CCl4 group (G2). In this regard, higher serum 25(OH)D levels might be associated with this finding, modulating hepatocyte cell proliferation in DEN/CCl4-treated groups. Cell proliferation plays an important role during critical phases of rat liver carcinogenesis, including the processes of initiation and promotion [50]. Therefore, the suppression of cell proliferation is considered an important feature for a chemopreventive candidate [51, 52].

Glutathione S-transferases (GST) comprise a group of phase II metabolic enzymes involved in cellular protection against xenobiotics, oxidative stress, and resistance against chemotherapeutic compounds [53, 54]. The rat GST-P 7-7, an isozyme of glutathione S-transferase, is abnormally expressed in the early stages of chemical hepatocarcinogenesis. Therefore, single hepatocytes expressing GST-P develop very early in carcinogen-treated rat liver and are considered suitable markers of preneoplastic lesions [55, 56]. The detection of GST-P-positive foci is an important tool for analyzing relevant carcinogenic or anti-carcinogenic responses during the initiation and promotion stages of rat liver carcinogenesis [57, 58]. It was found that VD3 supplementation reduced the number of larger GST-P-positive PNL (> 1.0 mm2) when compared to the positive control group (G2). However, the underlying mechanisms by which VD3 can suppress GST-P-positive PNL development still need to be clarified.

Active VD3 effects are mediated by VDR which regulates gene expression in the target tissues [49]. The VDR is a member of a superfamily of nuclear steroid hormone receptors which participates in VD3 biological functions, regulating calcium and phosphorus homeostasis, immune response, cell differentiation, and cell proliferation [58]. This nuclear receptor can be found in many type cells throughout the body, widely expressed in tissues such as intestines, lungs, kidneys, skin, bones, and liver [59]. Liver expression of VDR might be inversely associated with the severity of liver damage in a number of chronic diseases [27, 28]. Decreased VDR expression has been related to an increasing susceptibility to the development of carcinogen-induced cancers and may be considered a potential biomarker candidate for cancer prognostic [60, 61]. Many clinical trials have found evidence of reduced VDR expression in HCC and cholangiosarcoma alone but not in dysplastic nodules and hepatomas [27, 62]. For instance, in the present study, DEN/CCl4 regimen did not alter VDR protein expression in the experimental groups, indicating that low VDR expression is associated with the progression phase of liver disease rather than early hepatocarcinogenesis [63]. Besides, VD3 treatment did not increase VDR levels, which is consistent with a previous study indicating that dietary VD3 did not affect VDR gene expression in azoxymethane (AOM)-induced PNL in mice [64]. The results also showed that hepatic CYP27A1 protein expression was significantly higher in the DEN/CCl4-treated group (G2) than in the remaining groups (Fig. 5). Although mitochondrial CYP27A1 has been shown to activate 25-hydroxilate VD3, this enzyme has a relatively low affinity and plays a minor role in VD3 hydroxylation. CYP27A1 is a bifunctional enzyme also involved in bile acid and cholesterol metabolism [65]. Therefore, the increased CYP27A1 expression seen in the positive control group might be related to the CCl4-induced regimen that leads to mitochondrial dysfunction and oxidative metabolism impairment [31]. In fact, VD3 supplementation increased total glutathione levels and GSH-Px activity, as well as diminished lipid hydroperoxide levels in the liver [66], indicating a possible protective mechanism of VD3 against DEN/CCl4-induced hepatocarcinogenesis.

A limitation of this study is the short time of CCl4 exposure, which most likely did not allow observing the extensive liver lesions expected to occur with its use. Thus, studies including longer periods of CCl4 exposure are required to further investigate the molecular mechanisms and potential protective role of vitamin D against liver tumorigenesis.

Conclusions

Dietary factors have been in the spotlight of scientific interest since that they can exert preventive activities against human chronic diseases, including cancer [67]. It is currently known that vitamins play an important role in the prevention and treatment of precancerous and cancerous conditions [68], but until now, no conclusive results were obtained. Therefore, the findings the present study support the hypothesis that VD3 supplementation can reduce the early development of hepatocellular PNL in rats, but only in higher doses. However, the possibility of long-term VD3 high-dose toxicity should be investigated.

Declarations

Acknowledgements

This article is dedicated to the memory of professor Carlos Eduardo Andrade Chagas, acknowledging his significant and important contributions in this experimental study.

Funding

Mariana B. Tablas and Renata L. Goto were recipients of a fellowship from FAPESP (2012/03964-8) and CAPES (DS 2013-2016), respectively. Luis F. Barbisan was a recipient of support research from FAPESP (2012/03628-8).

Availability of data and materials

Please contact the author for data requests.

Authors’ contributions

All authors contributed equally to this work. All authors read and approved the final manuscript.

Ethics approval

This study was performed according to the Ethical Principles for Animals Research adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the Institution’s Ethics Review Board (403-CEUA).

Consent for publication

Not applicable.

Competing interests

All authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Department of Morphology, Institute of Biosciences, Sao Paulo State University, Botucatu, Brazil

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