Hesperidin

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

The aim of this study was to investigate the effect of hesperidin on male reproductive system in rats

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2016 - 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study did not follow OECD guidelines, but was planed to prove that compounds
with antioxidant properties such as hesperidin may protect testicular tissue from harmful effects of the oxidative stress caused by MTX. Further details see below.

GLP compliance:

yes

Remarks:

Asstudy was performed at a University, GLP compliance is anticipated; The study was approved by Yuzuncu Yıl University Local Ethics Committee On Animal Experiments (Approval number: 2015/28)

Test material

Test material information

Constituent 1

Reference substance name:

Hesperidin

EC Number:

208-288-1

EC Name:

Hesperidin

Cas Number:

520-26-3

Molecular formula:

C28H34O15

IUPAC Name:

5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside

Constituent 2

Reference substance name:

C28H34O15

IUPAC Name:

C28H34O15

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

The study was performed on 28 healthy male Wistar albino rats (8 week-old, with 250-300 g of body weight) which were provided from Fırat University Faculty of Medicine Experimental Research Center (Elazig, Turkey). Animals were adjusted to experimental conditions for a 1-week period before starting dosage. The animals were housed in standard laboratory conditions (24 ±3 °C temperature, 40 - 60% humidity and 12 h light/12 h darkness).
They were fed with commercial pelleted feed (Bayramoglu Food, Erzurum/Turkey) and fresh drinking water was provided ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

physiological saline

Duration of treatment / exposure:

hesperidin was administered for 7 days, 200 mg/kg bw/d.

Frequency of treatment:

7 days

Doses / concentrations

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

applied by gavage

No. of animals per sex per dose:

7

Control animals:

yes, concurrent no treatment

Positive control:

Group 2: methotrexate (MTX) group (n=7): A single dose of 20 mg/kg of MTX was administered I.P. Because, it was revealed in previous studies that this dose (20 mg/kg) causes tissue toxicity

Examinations

Statistics:

All statistical data were evaluated by using SPSS program (SPSS for windows, version 20.0). Spermatological and biochemical data were evaluated with Post hoc Tukey-HSD test and One-way variance analysis (ANOVA) in order to determine the differences between the groups. For histopathological and immunohistochemical findings, non-parametric Kruskal-Wallis and Mann-Whitney-U tests were used. P<0.05 value was accepted to be significant.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Spermatological Parameters: Sperm density, motility and abnormal sperm rate are given in Table 1. MTX group exhibited a significant decrease in sperm density and motility and a significant increase in abnormal sperm rate, compared to the control and other groups, whereas in the hesperidin group even a slight increase in sperm density and motility and a slight decrease in abnormal sperm rate was observed. In addition to this, it was determined that MTX with Hesperidin treatment reduced the increased abnormal sperm rate and increased the reduced sperm motility and sperm density, compared to the only MTX group (Table 1, P<0.05).
Biochemical Results: Biochemical results of the testicular tissue are given in Table 2. It was determined that in the testicular tissue, MDA level increased in MTX group compared to the control group and MTX + hesperidin administration lowered this level to a near value in the control group (P<0.05). The level in the hesperidin group was even lower than in the control (water) group 1. It was also determined that GSH level reduced in MTX group and MTX + hesperidin administration increased this lowered level (P<0.05). Hesperidin even increased the level compared to control.
It was determined that CAT, GPx and SOD activities were lower in the MTX-treated group compared to the control group, MTX + hesperidin administration led to an increase in enzyme activities and strengthened antioxidant defence system (P<0.05). Hesperidin itself even strengthened antioxidative defence compared to control.
When the testicular tissue was examined in terms of TNF-alpha and IL-1 Beta levels among cytokines, it was determined that the levels were significantly lower in the group for which hesperidin was administered alone, compared to the control group. With MTX administration, increase in TNF-alpha and IL-1 Beta levels occurred, and addition of hesperidin treatment to MTX treatment was observed to decrease TNF-alpha and IL-1 Beta levels (P<0.05).
Histopathological Findings: Structure of seminiferous tubules was observed to be normal in the control and hesperidin groups. It was recognized that in the group for which MTX was administered, regular structure of spermatogenic cells was impaired, and seminiferous tubules became necrotic and degenerative, with development of sporadic flaking. It was determined that in the group for which MTX was administered together with hesperidin, observed necrotic and degenerative changes were reduced compared to the group for which MTX was administered alone, P<0.05).
Immunohistochemical Findings: 8-OhDG expression was determined to be extremely low in control and hesperidin groups. It was determined that in the group for which MTX was administered alone, 8-OhDG was expressed strongly in seminiferous tubules but 8-OhDG expression level decreased in the group for which MTX was administered together with hesperidin (P<0.05). NFKB1 expression, however, was at extremely low level in control and hesperidin groups. Whereas it was observed that NFKB1 was expressed strongly in spermatids in the group for which MTX was administered alone, expression level was observed to begin decreasing in the group for which MTX was administered together with hesperidin (P<0.05)
DISCUSSION
Testicles are the most important target organs for oxidative stress due to their high content of polyunsaturated membrane lipids. Increased oxidative stress in testicles damages to spermatological parameters. In the current study, the decrease in sperm count and sperm motility and the increase in abnormal sperm rate determined following MTX administration confirmed previous studies. The increase in abnormal sperm rate and reduction in sperm density and motility are associated with the increased lipid peroxidation. This situation can be explained by that MTX damages cell membrane integrity by disturbing lipids and proteins within the sperm membrane. It was determined in previous studies that like MTX, Cisplatin causes a marked decrease in sperm density and motility.
Hesperidin administration in addition to MTX decreased effects of MTX on sperm parameters and increased sperm count and motility. Protective effects of hesperidin are probably may be referred to its obvious antioxidant potential which was observed in this study. Our biochemical findings indicated that MTX increased MDA level which is among the most important oxidant parameters in the testicular tissue, compared to the control group. This finding is consistent with some reports indicating that MTX stimulates oxidative stress by increasing MDA levels. Also, it revealed that MTX treatment significantly decreased endogenous antioxidant enzyme activities such as SOD, CAT and GPx and GSH level, which are commonly used for monitoring oxidant/antioxidant status. In the study, it was determined that Gpx, CAT and SOD enzyme activities, as well as GSH level, were significantly increased and lipid peroxidation induced by MTX was significantly reduced with hesperidin administration. These results are consistent with those of previous studies.
TNF-alpha exists in seminiferous tubules and is strongly up-regulated under both pathological and physiological conditions. IL-1 Beta is produced by macrophages. This cytokine is known as an important mediator of various cellular functions, including reproduction, differentiation and apoptosis, and of the inflammatory response. The increase in TNF-alpha and IL-1 Beta levels which was observed after MTX administration suggested that MTX led to an inflammatory reaction. Reduction of this increase in TNF-alpha and IL-1 Beta levels by administration of hesperidin in addition to MTX suggests that hesperidin has anti-inflammatory effects. It was reported in a previous study that MTX led to an increase in TNF-alpha level. It is consistent with this study that Cisplatin, Doxorubicin and Sodium nitrite increase TNF-alpha and IL-1 Beta levels among pro-inflammatory cytokines in testicular tissue.
In histopathological evaluation, severe necrotic and degenerative changes were determined in MTX group. In MTX + hesperidin group, however, necrotic and degenerative changes were determined to be milder. Therefore, possible protective effect of hesperidin was evaluated immuno-histochemically with 8-OhDG in regard to DNA damage and with NfKB in regard to inflammatory reaction. 8-OhDG is a form of free radicals and a biomarker used in oxidative stress. In various studies it was expressed that severity of DNA damage in testicular destruction due to oxidative stress was determined with 8-OhDG [44]. In the present study, whereas 8-OhDG was strongly expressed in MTX group, reduction in 8-OhDG expression in the group for which MTX was administered together with hesperidin suggested that DNA damage was diminished and, hence, hesperidin had a protective property. NfKB, however, is a molecule which is activated when oxidative stress is developed. It was reported that level of NfKB increased with testicular intoxication. Present study, NfKB was determined to be severe in MTX group and moderate in MTX + hesperidin group. This situation was expressed as that hesperidin reduced inflammatory reaction. This study demonstrated that administration of a single dose of 20 mg/kg of MTX increased Iipid peroxidation levels in testicles of Wistar albino rats and, thus, caused oxidative stress. Additionally, it also demonstrated that it had toxic effects including histopathological changes and spermatological damage. Treatment of MTX together with hesperidin was determined to significantly prevent toxicity of MTX on reproductive system. When results of all groups are taken into consideration, we suggest that hesperidin has a regenerative effect on testicular tissue and sperm parameters.
Hesperidin as such when compared to the control (water in this study) did not show any negative effects on any of the parameters observed. Quite contrary, even compared to control the animals in the hesperidin group showed even slightly positive developments on some parameters.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

hesperidin

Sex:

male

Basis for effect level:

other: all parameters observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

Effect levels (F1)

Remarks on result:

not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

No negative effects in any of the investigated parameters (spermatological parameters, biochemical results, histopathology, immunohistochemical parameters) were seen in the hesperidin dose group, dosed at 200 mg/kg bw/day for 7 days..

Executive summary:

The aim of this study was to investigate the effect of hesperidin on male reproductive system in rats to which methotrexate (MTX) was administered. In the study, 28 male Wistar albino rats at the age of 8 weeks and had 250-300 g of live weight were used. Four experimental groups were formed; Group 1 (n=7): The control group, only feed and water were given. Group 2 (n=7): MTX group, a single dose of 20 mg/kg of i.p. MTX was administered. Group 3 (n=7): Hesperidin group, 200 mg/kg of hesperidin was administered by gavage for 7 days. Group 4: MTX + hesperidin group (n=7): Following administration of a single dose of 20 mg/kg i.p. MTX , 200 mg/kg of Hesperidin was administered by oral gavage for 7 days. At the end of the experiment, rats were decapitated and biochemical, histopathological and spermatological parameters were examined. It was observed that in the MTX group, sperm motility and density, the enzymes CAT, GPx and SOD and GSH level decreased, TNF-alpha and IL-1 Beta, as well as MDA, levels were increased, regular structure of spermatogenic cells was impaired, and seminiferous tubules became necrotic and degenerative. It was determined that spermatological parameters improved and, necrotic and degenerative changes diminished by the administration of MTX+hesperidin. These outcomes indicated that hesperidin had a protective effect on destructive effects of MTX in rat testicles. No negative effects in any of the investigated parameters were seen in the hesperidin dose group.