Comparative Toxicology of Heavy Metals Arsenate, Arsenite, Cadmium, Chrome Vi, Lead, and Nickel in the Testes of Adult Swiss Mice After Acute Exposure

doi:10.21203/rs.3.rs-1949599/v1

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Comparative Toxicology of Heavy Metals Arsenate, Arsenite, Cadmium, Chrome Vi, Lead, and Nickel in the Testes of Adult Swiss Mice After Acute Exposure

https://doi.org/10.21203/rs.3.rs-1949599/v1

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Heavy metals are found naturally in the environment in small concentrations and the increase in their concentration occurs due to anthropogenic activities. Human exposure to heavy metals can induce various testicular damage. This study aimed to evaluate the action of the arsenate (As ^+ 5), arsenite (As ^+ 3), cadmium (Cd), lead (Pb), chromium VI (Cr VI), and nickel (Ni) in the testicles of adult Swiss mice and establish an order of toxicity among these metals. Forty-two animals were randomly distributed into 7 experimental groups (n = 6). The control group received a saline solution and the other groups received 1.5 mg/kg of the heavy metal as follows: group 2 – As ^+ 5, group 3 - As ^+ 3, group 4 - Cd, group 5 - Pb, group 6 - Cr VI and group 7 - Ni. The administration was performed intraperitoneally in a single dose and the animals were euthanized seven days after exposure. Acute exposure to heavy metals induced functional, cellular, pathological, and oxidative stress alterations. The order of toxicity among the tested metals established by comparative damage analysis follows Ni > Cd > Cr VI = Pb > As ^+ 3 > As ^+ 5. In groups with higher toxicity, such as nickel and cadmium were observed functional changes, alterations in the testicular microstructure, and cell death. Based on our results, we can point out that the heavy metals tested can be harmful to male reproductive health in low doses and for a short period of exposure. Care must be greater taking into account the damage order defined in this work.

Environmental contaminants

Heavy metals

Reproduction

Testicle

In recent decades, human exposure to environmental heavy metals has become more common [1]. This fact led to several studies in order to identify the emergence of problems resulting from this exposure [1]. Certain metals, in moderate concentrations, are essential for maintaining the body's physiological functions [2, 3, 1]. However, non-essential metals, generally known as heavy metals, have no established biological role and are toxic to the body [2, 3]. Heavy metals are stable chemicals that in contact with living beings can cause adverse physiological changes [4, 5]. Heavy metals are found naturally in the environment in small concentrations and the increase in their concentration occurs due to anthropogenic activities [4, 5].

There are several studies reporting the action of heavy metals in the testicles showing the ability of these compounds to interfere in the testicular function and decrease the reproductive capacity [6, 1, 7]. Changes such as reduced testicular weight, histological and hormonal changes, seminiferous tubules degeneration, apoptosis and necrosis have also been reported [8, 9, 10, 6, 7]. Exposure to heavy metals can also induce testicular oxidative stress by decreasing sperm quality which is a major cause of infertility [11, 12].

Knowing the action of heavy metals in the testicles such as arsenate, arsenite, cadmium, lead, chromium VI, and nickel, which are found with great frequency in the environment, is important to define their degree of danger for reproductive success. From the obtained information it is possible to establish an order of toxicity and define means of decontamination as well as the disposal of these heavy metals. Therefore, the aim of the present study is to evaluate the action of the heavy metal’s arsenate, arsenite, cadmium, lead, chromium VI and nickel in the testicles of adult Swiss mice. To establish an order of testicular toxicity among these metals, biometric, morphometric, stereological, oxidative stress and histopathological analysis were made.

Animals

Forty-two male Swiss mice (40g; 70 days old) were randomly maintained under controlled photoperiod (12-12h light/dark), humidity (60–70%) and temperature (22 ± 2ºC). The animals were kept for 15 days during the acclimatization period before the beginning of the experiment and had access to water and food ad libitum. All the experimental procedures followed the standards determined by National Council for the Control of Animal Experimentation and were approved by the Ethics Committee of Animal Use of the Federal University of Viçosa (protocol 07/2018).

Chemicals

Arsenic (Sigma) was used in the form of sodium arsenite dibasic heptahydrate (Na₂HAsO₄.7H₂O) and sodium arsenate (NaAsO₂). Were used cadmium chloride (CdCl₂; Vetec), lead acetate trihydrate (Pb(C₂H₃O₂)₂.3H₂O; Vetec), chromium VI trioxide (CrO₃; Sigma) and nickel (II) chloride hexahydrate (NiCl₂.6H₂O; Sigma). The salts were dissolved separately in distilled water in their respective doses to obtain 1.5 mg/kg of body weight of the heavy metal. The administration was made in the amount of 0.7 ml of solution per animal.

Experimental design and tissue collection

After 15 days of acclimation, the animals (n = 42) were randomly distributed into 7 experimental groups (n = 6) and kept in collective polypropylene cages. The control group (group 1) received 0.7 ml of saline solution (0.9%) and the other groups received 0.7 ml of salt solution (1.5 mg/kg of the heavy metal) as following: group 2 - arsenate (6.24 mg/kg of salt), group 3 - arsenite (2.60 mg/kg of salt), group 4 - cadmium (2.44 mg/kg of salt), group 5 - lead (2.74 mg/kg of salt), group 6 - chromium VI (2.88 mg/kg of salt) and group 7 - nickel (6.07 mg/kg of salt). The administration was performed intraperitoneally in a single dose (i.p. SD), and the animals were euthanized seven days after exposure. The dosage of 1.5 mg/kg was based on previous studies [6, 7]. The 7-day period was defined to observe probable changes caused by an acute exposure, before a possible recovery process could alter the degree of damage caused by the metals [13].

On the eighth day of the experiment, the animals were weighed, anesthetized with sodium thiopental (30 mg/kg i.p.), and euthanized by deepening anesthesia (sodium thiopental, 150 mg/kg i.p.), followed by cardiac puncture and exsanguination. At the time of euthanasia, blood was collected from each animal into heparinized tubes, stored, and subsequently centrifuged for plasma separation (2000 rpm/15 min) which was frozen at − 20 ◦C until testosterone concentrations were quantified.

The testicles were removed and weighed. The left testicles were fixed in Karnovsky [14] for histological evaluation and dosage of heavy metals. After 24 h of fixation, the testicles were transferred to 70% ethanol, and testicular fragments were dehydrated in ethanol and embedded in glycolmethacrylate (Historesin®, Leica Microsystems, Nussloch, Germany). Semi-serial histological sections (3 µm thick) were made using a rotary microtome (RM 2255, Leica Microsystems, Nussloch, Germany), with a minimum of 40 µm between sections, and stained with toluidine blue/sodium borate 1%. The digital images were obtained using a light-field photomicroscope (Olympus BX-53, Tokyo, Japan) connected to a digital camera (Olympus AX 70 TRF, Tokyo, Japan), and analyzed using the ImageJ® (National Institute of Health, USA) software. The right testicles were frozen to -20^oC for oxidative status analyses.

Biometric parameters

After the fixation period, the tunica albuginea of the left testicle from each animal was removed, weighed, and its weight was subtracted from the total testicle weight, providing the testicular parenchyma weight. Based on the body and testicular weights, the gonadosomatic index (GSI) was obtained by dividing the testicular weight by body weight and multiplied by 100 [15]. The parenchymosomatic index (PSI) was determined by dividing the testicular parenchyma weight by body weight and multiplied by 100.

Tubular morphometry

The volumetric density of all seminiferous tubule's components (tunica propria, seminiferous epithelium and lumen) was determined after counting 5320 intersection points, per animal, in 20 square grids (266 intersections/points each) placed randomly over digital images. The volume of each component was estimated from the knowledge of its percentage within the testis and the knowledge of the testicular parenchyma volume. Since the mammalian testis density is around 1 [16], its weight was considered as the same as the volume. The tubulesomatic (TSI) and epithelium-somatic (ESI) indexes were calculated based upon the body weight and the tubular and epithelial volumes, respectively (tubular or epithelial volume/body weight x 100).

The mean tubular diameter was obtained after measuring 20 random circular seminiferous tubules cross sections from each animal, regardless the tubular stage. The seminiferous epithelium height was measured in the same tubular sections in which the tubule diameter was obtained (as the mean of two diametrically opposed measurements). The luminal diameter was obtained by subtracting the tubular diameter from the seminiferous epithelium height. The tubular (STAr), luminal (LAr) and epithelial (EAr) areas were calculated as following: STAr = πTR², where TR = tubular radius; LAr=.πLR², here LR = luminal radius; EAr = STAr – LAr. The tubule epithelium ratio (TER) was calculated by dividing STAr/EAr.

The seminiferous tubules length was estimated from the knowledge of the volume occupied by them in the testicles and the mean tubular diameter obtained for each animal [17]. The obtained results were divided by the testicular weight in order to calculate the seminiferous tubules length per gram of testis.

Cell counting

The estimation of the germ and Sertoli cells number was carried out in seminiferous epithelium in stage 1 of the seminiferous epithelium cycle [18]. Type A spermatogonia (A), primary spermatocyte at preleptotene/leptotene (PL/L), primary spermatocyte at pachytene (P), round spermatid (RS) and Sertoli cell (S) were quantified in 20 random tubular cross sections per animal. Thirty nuclear diameters of each germ cell type and nucleolar diameters of the Sertoli cells were measured for each animal. The results were corrected due to variations in the size of the cells and the section thickness as described by Amann and Almquist (1962) [19]. The following indexes were calculated from the corrected numbers: spermatogonial mitosis (PL/L: A), meiotic index (RS: P), spermatogenic yield (RS: A), Sertoli cell index (RS: S), and the total support capacity of the Sertoli cell (A + PL/L + P + RS: S).

The number of Sertoli cells (SC) was determined as following: SC = (STL x S)/3 (STL = seminiferous tubules length; S = Sertoli cells number; 3 = section thickness). The obtained results were divided by the testicular weight in order to calculate the number of Sertoli cells per gram of testis (SC/g).

Daily spermatic production was calculated according to Amann and Almquist (1962) [19]. These values were divided by the testicular weight in order to calculate the daily spermatic production per gram of testis.

Intertubular morphometry

The volumetric density of intertubular components (nucleus and cytoplasm of Leydig cell, blood vessels, lymphatic space, macrophages, and connective tissue) was determined by counting 1000 intersection points, per animal, over intertubular images. The volume (mL) of each component was calculated by multiplying the volumetric density of the element in the testicles by the testicular parenchyma weight and divided by 100.

The mean diameter of the Leydig cell nucleus was obtained by measuring 30 nuclei, per animal, showing characteristic perinuclear chromatin and evident nucleoli. The nuclear volume was obtained as following: nuclear volume = 4/3 πR³, where R = nuclear diameter/2. The cytoplasmic volume was estimated by multiplying the percentage of cytoplasm by the nuclear volume, divided by the nuclear percentage. The single cell volume was calculated by adding the nuclear and cytoplasmic volumes.

The volume that the Leydig cells occupy in the testes was calculated by multiplying the volume density of Leydig cells in the testicular parenchyma by the parenchyma weight of the testes and divided by 100. The obtained results were divided by the testicular weight in order to calculate the volume that the Leydig cells occupy per gram of testis.

The number of Leydig cells in the testicles was calculated from the Leydig cell individual volume and the total volume occupied by Leydig cells in the testicular parenchyma. To calculate the number of Leydig cells per gram of testis, the volume that the Leydig cell occupies per gram of testis was divided by the volume of a Leydig cell. The Leydigosomatic index (LSI), which quantifies the energy investment in Leydig cells related to body mass, was obtained by dividing the total volume of Leydig cell in the testicular parenchyma by the body weight and multiplied by 100.

Testosterone concentration

The serum testosterone concentration was obtained by chemiluminescence by using Access® Testosterone kit (Beckman Coulter, Inc., California, USA). The results were expressed in ng/dL.

Histopathology

The proportions of normal and pathological seminiferous tubules were estimated by counting 200 random tubules in histological preparations of the testis of each animal. Tubules with pathologies were considered those with morphological alterations [20, 21]. According to Johnsen (1970) [22] there are 10 alterations levels, but in this study was used the Johnsen's score modified for 8 levels: Level 1 - intact tubules with germ cells arranged at their normal localization site and few vacuoles; Level 2 – vacuoles at the base of the epithelium; Level 3 – vacuoles at the apex of the epithelium; Level 4 – vacuoles at the apex and base of the epithelium; Level 5 - spermatogenic cells in the tubular lumen and cell degeneration; Level 6 – tubules with only basal cells; Level 7 – tubules with only Sertoli cells; Level 8 – tubules with no Sertoli or germ cells, characterizing an irreversible state of degeneration. These levels were grouped into normal (Level 1), slight pathologies (Levels 2 and 3), moderate pathologies (Levels 4 and 5) and severe pathologies (Levels 6, 7 and 8) [23].

Cell availability

Histological sections of 1 µm were stained with acridine orange (AO) and propidium iodide (PI) for evaluating cellular morphological alterations [24]. Acridine orange is a vital dye that stains both live (or viable cells) and dead cells (or apoptotic cells), whereas propidium iodide stains only cells that have lost their membrane integrity. Viable cells exhibit a green nucleus while non-viable cells exhibit a dense red nucleus. The digital images were captured using the fluorescence photomicroscope EVOS fl (Life Technologies, Carlsbad, Canada). The scanned images were sent to ImageJ® software and areas with double marking (AO/PI) were selected by color distribution as discriminant parameter. After the definition of the color interval by means of trial and error, the area representing the amount of cells in initial damage process (yellow/orange) or dead cells (red) was selected in each field. After, the mask was used to provide the area and percentage of damaged or dead germ cells or sperm in each field. A total area of 30 × 104 µm² was quantified for each group [25].

Heavy metals bioaccumulation on testicular tissue

Samples of the left testis were weighed and dried at 70 ° C until reaching constant dry weight. The dried samples were placed in Erlenmeyer flasks with 1.5 mL of concentrated nitric acid (HNO₃) and 0.5 mL of perchloric acid (HClO₄, 70%) and transferred to the hot plate. The temperature was gradually raised to 90°C so that the digestion of the material was complete. The samples were diluted in deionized water and filtered. The concentrations of metals were determined using an atomic absorption spectrophotometer (SpectrAA 220FS Varian).

Oxidative status analyses

The frozen testes were homogenized in potassium phosphate buffered saline (pH 7.4, 0.2 M) containing 1 M EDTA and centrifuged (1200 rpm, 4 ° C, for 10 min). The supernatant was used for the analyses, which were performed in duplicate.

The activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were evaluated. SOD activity was determined according to the Sarban et al. (2005) [26] protocol. CAT activity was evaluated by measuring the decomposition rate of hydrogen peroxide (H₂O₂) [27]. GST activity was measured through the formation of 1-chloro-2,4-dinitrobenzene conjugate (CDNB) [28].

Oxidative stress markers such as malondialdehyde (MDA) and nitric oxide (NO) were also evaluated. For the MDA analysis, the homogenate reacted with thiobarbituric acid reactive substance solution (TBARS; 15% trichloroacetic acid, 0.375% thiobarbituric acid and HCl 0.25 N) for 40 min in water bath at 90°C. TBARS formation was monitored at 535 nm in a microplate spectrophotometer (Power Wave X) [29]. NO production was quantified indirectly through the nitrite/nitrate content by the standard Griess reaction [30]. The homogenate supernatant was incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine hydrochloride, and 2.5% H₃PO₄), at room temperature, for 10 min. The absorbance was measured at 570 nm, in a microplate spectrophotometer (Power Wave X) and the nitrite concentration was calculated with reference to the standard curve (0.0610 to 125 µM) of sodium nitrite (NaNO₂).

Statistical analysis

The results were submitted to a normality test (Shapiro-Wilk). The data were submitted to analysis of variance (ANOVA) followed by Student Newman-Keuls test comparing treatments with the control group. All statistical analyses were made using the Statistics Software, and the results were reported as mean ± standard deviation of the mean. Statistical significance was set at p < 0.05.

All summarized results can be found in the Tables S1, S2 and S3.

Biometric parameters

There were no changes in the biometric and testicular parameters, as well as in the gonadosomatic and parenchymosomatic indexes between the experimental groups compared to the control (Table 1).

Table 1

– Biometric and testicular parameters of male Swiss mice after acute exposure of 1.5 mg/kg Arsenate, Arsenite, Cadmium, Lead, Chromium VI and Nickel.
Parameters	Control	Arsenate	Arsenite	Cadmium	Lead	Cromium VI	Nickel
Body weight (g)	39.09 ± 2.81	39.54 ± 2.31	38.07 ± 3.20	36.84 ± 2.15	37.45 ± 2.32	38.36 ± 1.62	35.84 ± 2.89
Testicular weight (g)	0.21 ± 0.03	0.22 ± 0.02	0.22 ± 0.02	0.23 ± 0.02	0.22 ± 0.02	0.21 ± 0.02	0.21 ± 0.02
Albuginea weight (g)	0.02 ± 0.01	0.01 ± 0.00	0.03 ± 0.01	0.01 ± 0.00	0.03 ± 0.01	0.02 ± 0.01	0.03 ± 0.01
Parenchyma weight (g)	0.19 ± 0.02	0.21 ± 0.02	0.19 ± 0.02	0.22 ± 0.02	0.19 ± 0.02	0.19 ± 0.02	0.18 ± 0.02
Gonadosomatic index (%)	0.53 ± 0.08	0.55 ± 0.02	0.59 ± 0.08	0.62 ± 0.08	0.59 ± 0.09	0.54 ± 0.05	0.58 ± 0.08
Parenchymosomatic index (%)	0.48 ± 0.07	0.53 ± 0.03	0.51 ± 0.07	0.61 ± 0.09	0.51 ± 0.07	0.49 ± 0.06	0.50 ± 0.07

Data are expressed as mean ± standard deviation of the mean (n = 6). (*) indicate significant change from control at p ≤ 0.05 (Student Newman Keuls test).

Tubular morphometry

Table 2 contains the volumetric density and volume of tubular components, as well as tubulesomatic and epitheliumsomatic indexes.

The percentage of seminiferous epithelium decreased in cadmium, lead, chromium VI and nickel groups in relation to the control group, accompanied by a significant increase in the percentage and volume of tunica propria. The other parameters analyzed did not show any significant difference in the treated groups in relation to the control.

The stereology of seminiferous tubules did not show significant difference in the treated groups in relation to the control (Table 3).

Cell counting

Data referring to the analyzes performed in the seminiferous tubules in stage 1 of the seminiferous epithelium cycle are presented in Table 4. The corrected number of germ and Sertoli cells, ratios between cell types, Sertoli cells number and daily spermatic production remained unchanged in all experimental groups compared to the control group.

Table 2

– Volumetric density (%) and volume (µL) of tubular components, and tubulesomatic and epithelium-somatic indexes of Swiss mice after acute exposure of 1.5mg/kg Arsenate, Arsenite, Cadmium, Lead, Chromium VI and Nickel.
Parameters	Control	Arsenate	Arsenite	Cadmium	Lead	Cromium VI	Nickel
Seminiferous tubule (%)	91.76 ± 1.37	91.07 ± 1.20	91.38 ± 1.49	91.09 ± 1.75	91.44 ± 1.23	90.44 ± 1.74	90.96 ± 1.88
Seminiferous epithelium (%)	79.12 ± 0.25	79.60 ± 1.53	76.58 ± 1.42	76.13 ± 1.81 *	75.01 ± 1.48 *	75.26 ± 2.18 *	75.21 ± 2.02 *
Tunica propria (%)	4.87 ± 0.67	5.23 ± 0.25	5.82 ± 1.06	7.58 ± 0.55 *	7.57 ± 0.92 *	7.68 ± 0.55 *	7.58 ± 0.45 *
Lumen (%)	7.77 ± 1.99	7.24 ± 1.50	8.98 ± 2.00	7.38 ± 1.13	8.85 ± 1.42	7.50 ± 1.37	8.18 ± 1.77
Seminiferous tubule (µL)	172 ± 21.74	192 ± 17.30	177 ± 18.46	202 ± 25.06	175 ± 16.30	170 ± 22.35	162 ± 17.50
Seminiferous epithelium (µL)	148 ± 21.25	165 ± 16.50	149 ± 14.54	169 ± 25.02	144 ± 13.25	142 ± 19.13	134 ± 15.95
Tunica propria (µL)	9.19 ± 2.02	11.04 ± 1.45	11 ± 1.88	17 ± 2.61 *	14 ± 1.36 *	14 ± 1.36 *	14 ± 1.84 *
Lumen (µL)	14.27 ± 2.77	15.08 ± 2.36	18 ± 5.09	16 ± 3.42	14 ± 3.56	14 ± 3.27	14 ± 2.46
Tubulesomatic index (%)	0.44 ± 0.07	0.48 ± 0.02	0.47 ± 0.07	0.55 ± 0.08	0.47 ± 0.06	0.45 ± 0.06	0.45 ± 0.07
Epithelium-somatic index (%)	0.38 ± 0.06	0.42 ± 0.02	0.39 ± 0.06	0.46 ± 0.07	0.39 ± 0.05	0.37 ± 0.06	0.38 ± 0.06

Data are expressed as mean ± standard deviation of the mean (n = 6). (*) indicate significant change from control at p ≤ 0.05 (Student Newman Keuls test).

Table 3

– Stereology of seminiferous tubules of Swiss mice after acute exposure of 1.5mg/kg of Arsenate, Arsenite, Cadmium, Lead, Chromium VI and Nickel.
Parameters	Control	Arsenate	Arsenite	Cadmium	Lead	Cromium VI	Nickel
Tubular diameter (µm)	197.52 ± 8.60	198.31 ± 7.96	203.39 ± 5.46	200.66 ± 9.23	200.79 ± 13.15	194.19 ± 6.93	196.52 ± 7.32
Epithelium height (µm)	79.01 ± 3.79	78.96 ± 4.09	78.47 ± 5.01	79.82 ± 4.07	80.19 ± 3.29	79.65 ± 2.44	78.55 ± 2.83
Lumen diameter (µm)	39.51 ± 8.53	40.38 ± 6.4	46.45 ± 9.33	41.02 ± 13.06	40.40 ± 8.45	34.88 ± 5.60	39.42 ± 11.34
STL (m)	5.66 ± 1.02	6.26 ± 1.05	5.46 ± 0.52	6.42 ± 0.84	5.57 ± 0.62	5.80 ± 1.04	5.38 ± 0.93
STL/g (m/g)	27.33 ± 2.84	28.71 ± 2.76	24.36 ± 1.18	28.14 ± 3.00	25.61 ± 3.94	27.64 ± 3.11	29.18 ± 3.31
STAr (µm²x10⁴)	3.07 ± 0.27	3.09 ± 0.25	3.25 ± 0.17	3.17 ± 0.28	3.18 ± 0.40	2.96 ± 0.21	3.04 ± 0.23
LAr (µm²x10⁴)	0.13 ± 0.05	0.13 ± 0.04	0.18 ± 0.07	0.14 ± 0.09	0.13 ± 0.05	0.10 ± 0.03	0.13 ± 0.08
EAr (µm²x10⁴)	2.94 ± 0.24	2.96 ± 0.23	3.07 ± 0.18	3.02 ± 0.23	3.04 ± 0.36	2.87 ± 0.19	2.90 ± 0.16
Tubule/Epithelium ratio	1.04 ± 0.02	1.04 ± 0.01	1.06 ± 0.02	1.05 ± 0.03	1.04 ± 0.01	1.03 ± 0.01	1.04 ± 0.03
STL = seminiferous tubules length; STL/g = seminiferous tubules length per gram of testis; STAr = seminiferous tubule area; LAr = lumen area; EAr = epithelium area. Data are expressed as mean ± standard deviation of the mean (n = 6). (*) indicate significant change from control at p ≤ 0.05 (Student Newman Keuls test).

Table 4

Corrected number of cells in stage 1 of the seminiferous epithelium cycle, ratios between cell types, Sertoli cells number and daily spermatic production of male Swiss mice after acute exposure of 1.5 mg/kg Arsenate, Arsenite, Cadmium, Lead, Chromium VI and Nickel.
	Control	Arsenate	Arsenite	Cadmium	Lead	Cromium VI	Nickel
Spermatogonia	2,02 ± 0,54	2,02 ± 0,54	1,86 ± 0,49	2,46 ± 0,40	1,96 ± 0,19	1,84 ± 0,29	1,82 ± 0,54
Preleptotene/leptotene	52,24 ± 4,76	53,10 ± 3,71	56,26 ± 5,03	55,32 ± 2,64	55,28 ± 5,15	52,04 ± 2,90	56,90 ± 5,83
Pachytene	65,94 ± 6,03	82,00 ± 34,00	74,36 ± 9,50	83,76 ± 32,15	73,88 ± 7,04	70,84 ± 3,05	71,04 ± 5,26
Round spermatid	126,10 ± 11,16	118,36 ± 10,50	129,72 ± 6,66	122,30 ± 5,92	120,20 ± 9,47	113,70 ± 6,46	115,30 ± 6,59
Sertoli cell	6,36 ± 3,71	7,96 ± 0,94	10,36 ± 4,03	8,62 ± 1,17	8,22 ± 1,23	8,64 ± 0,59	8,06 ± 1,07
Mitotic index	28,96 ± 9,12	29,94 ± 8,39	33,31 ± 7,62	24,02 ± 3,27	29,42 ± 3,95	30,11 ± 4,41	35,81 ± 11,63
Meiotic index	2,44 ± 0,14	2,01 ± 0,52	2,22 ± 019	2,02 ± 0,52	2,07 ± 0,15	2,03 ± 0,20	2,06 ± 0,14
Sertoli Cell index	13,10 ± 4,98	11,25 ± 2,23	10,17 ± 2,66	10,64 ± 1,28	10,85 ± 1,54	9,45 ± 0,14	10,29 ± 0,89
Number of Sertoli cells (x10^− 5)	0,88 ± 0,50	1,35 ± 0,35	1,49 ± 0,67	1,47 ± 0,34	1,15 ± 0,16	1,35 ± 0,20	1,15 ± 0,18
Number of Sertoli cells/gram of testis (x10^− 5)	4,38 ± 2,56	6,07 ± 1,28	6,47 ± 2,31	6,43 ± 0,98	5,46 ± 0,82	6,42 ± 0,56	5,70 ± 0,70
Support capacity of Sertoli cells	23,75 ± 8,91	21,95 ± 3,74	18,88 ± 5,01	20,77 ± 1,94	20,76 ± 3,01	18,18 ± 0,76	20,06 ± 1,84
Spermatogenic yield	66,38 ± 10,58	74,21 ± 24,40	86,40 ± 22,32	58,44 ± 6,20	71,52 ± 9,28	73,01 ± 12,47	80,50 ± 27,95
DTSP (x10⁶)	15,41 ± 3,47	16,52 ± 3,33	15,26 ± 1,21	17,17 ± 2,70	13,69 ± 0,80	14,19 ± 2,49	13,12 ± 2,04
DTSP/gt (x10⁶)	72,86 ± 8,03	74,05 ± 10,11	67,85 ± 3,16	75,46 ± 10,58	65,10 ± 8,56	67,42 ± 6,82	64,91 ± 7,54
DSP – Daily spermatic production; DSP/gt – Daily spermatic production per gram of testis. Data are expressed as mean ± standard deviation of the mean (n = 6). (*) indicate significant change from control at p ≤ 0.05 (Student Newman Keuls test).

Intertubular morphometry

The volumetric density and volume of intertubular components are shown in Table 5. The analyzed parameters were not altered in experimental groups.

The Leydig cell morphometry and stereology are shown in Table 6. The group treated with arsenite showed a reduction in Leydig nuclear diameter, nuclear, cytoplasmic and Leydig cell volumes, as well as an increase in the number of Leydig cells in the testes and per gram of testis in relation to the control group. Groups treated with lead, chromium VI and nickel showed an increase in Leydig nuclear diameter and nuclear volume compared to the control.

Table 5

– Volumetric density (%) and volume (µL) of intertubular components of male Swiss mice after acute exposure of 1.5 mg/kg Arsenate, Arsenite, Cadmium, Lead, Chromium VI and Nickel.
Parameters	Control	Arsenate	Arsenite	Cadmium	Lead	Cromium VI	Nickel
Intertubule (%)	8,24 ± 1,37	8,93 ± 1,20	8,62 ± 1,49	8,91 ± 1,80	9,56 ± 1,74	8,56 ± 1,23	9,04 ± 1,88
Blood vessel (%)	0,43 ± 0,22	0,49 ± 0,20	0,57 ± 0,12	0,63 ± 0,23	0,67 ± 0,20	0,58 ± 0,22	0,47 ± 0,20
Lymphatic space (%)	1,22 ± 0,79	1,34 ± 0,99	0,64 ± 0,26	0,95 ± 0,88	1,12 ± 0,77	0,73 ± 0,85	0,88 ± 0,64
Leydig nucleus (%)	0,95 ± 0,27	1,21 ± 0,21	1,37 ± 0,34	1,15 ± 0,21	1,46 ± 0,48	1,20 ± 0,21	1,44 ± 0,43
Leydig's cytoplasm (%)	5,44 ± 0,97	5,67 ± 0,69	5,77 ± 1,14	6,04 ± 1,59	6,20 ± 1,25	5,91 ± 1,05	6,03 ± 1,62
Leydig cell (%)	6,39 ± 1,19	6,88 ± 0,84	7,14 ± 1,39	7,19 ± 1,77	7,66 ± 1,67	7,11 ± 1,23	7,47 ± 2,02
Connective tissue (%)	0,19 ± 0,13	0,22 ± 0,09	0,27 ± 0,20	0,13 ± 0,03	0,11 ± 0,05	0,14 ± 0,06	0,22 ± 0,11
Macrophage (%)	0,00 ± 0,01	0,00 ± 0,00	0,00 ± 0,00	0,00 ± 0,00	0,00 ± 0,00	0,00 ± 0,00	0,00 ± 0,00
Intertubule (µL)	15,34 ± 2,74	18,83 ± 3,33	16,56 ± 2,07	19,56 ± 3,15	16,44 ± 2,94	17,73 ± 2,94	16,00 ± 2,04
Blood vessel (µL)	0,79 ± 0,40	1,05 ± 0,48	1,10 ± 0,20	1,36 ± 0,40	1,13 ± 0,46	1,24 ± 0,31	0,85 ± 0,40
Lymphatic space (µL)	2,31 ± 1,48	2,92 ± 2,32	1,25 ± 0,52	2,10 ± 1,84	1,44 ± 1,68	2,05 ± 1,29	1,52 ± 1,04
Leydig nucleus (µL)	1,75 ± 0,44	2,54 ± 0,46	2,63 ± 0,52	2,55 ± 0,45	2,29 ± 0,40	2,68 ± 0,71	2,55 ± 0,79
Leydig cytoplasm (µL)	10,13 ± 1,83	11,86 ± 1,24	11,10 ± 1,90	13,25 ± 2,98	11,32 ± 2,19	11,55 ± 2,06	10,69 ± 2,93
Leydig cell (µL)	11,88 ± 2,10	14,40 ± 1,55	13,73 ± 2,19	15,80 ± 3,34	13,61 ± 2,53	14,23 ± 2,54	13,24 ± 3,68
Connective tissue (µL)	0,35 ± 0,23	0,46 ± 0,18	0,49 ± 0,31	0,30 ± 0,09	0,27 ± 0,12	0,21 ± 0,10	0,38 ± 0,18
Macrophage (µL)	0,000 ± 0,01	0,000 ± 0,00	0,000 ± 0,00	0,000 ± 0,00	0,000 ± 0,00	0,000 ± 0,00	0,000 ± 0,00

Data are expressed as mean ± standard deviation of the mean (n = 6). (*) indicate significant change from control at p ≤ 0.05 (Student Newman Keuls test).

Table 6

– Leydig cell morphometry and stereology of male Swiss mice after acute exposure of 1.5 mg/kg Arsenate, Arsenite, Cadmium, Lead, Chromium VI and Nickel.
Parameters	Control	Arsenate	Arsenite	Cadmium	Lead	Cromium VI	Nickel
Nuclear Diameter (µm³)	7,35 ± 0,36	7,48 ± 0,041	6,78 ± 0,28 *	7,48 ± 0,31	8,30 ± 0,39 *	8,09 ± 0,24 *	8,21 ± 0,28 *
Nuclear volume (µm³)	209,48 ± 30,99	220,85 ± 36,47	164,11 ± 20,25 *	220,03 ± 27,75	300,71 ± 45,11 *	277,50 ± 24,19 *	290,50 ± 30,28 *
Cytoplasmic volume (x10²) (µm³)	12,40 ± 2,82	10,46 ± 1,76	7,07 ± 1,63 *	11,47 ± 2,17	13,38 ± 3,34	13,74 ± 1,63	12,38 ± 2,27
Cell volume (x10²) (µm³)	14,48 ± 2,95	12,67 ± 1,99	8,71 ± 1,75 *	13,67 ± 2,67	16,39 ± 3,53	16,51 ± 1,72	15,28 ± 2,51
Leydig volume (x10⁹) (µm³)	11,88 ± 2,10	14,40 ± 1,55	13,73 ± 2,19	11,88 ± 2,10	14,23 ± 2,54	13,61 ± 2,53	13,24 ± 3,68
Leydig volume /gt (x10⁹) (µm³)	58,03 ± 11,30	66,59 ± 7,88	61,75 ± 11,81	69,69 ± 16,50	68,76 ± 14,23	62,25 ± 10,53	64,05 ± 13,96
Number of cells (x10⁶) (µm³)	8,47 ± 2,17	11,63 ± 2,36	16,22 ± 3,69 *	11,66 ± 22,26	8,95 ± 2,37	8,21 ± 0,94	8,80 ± 2,56
Number of cells /gt (x10⁶) (µm³)	42,03 ± 13,50	53,38 ± 8,21	72,74 ± 17,27 *	51,14 ± 9,22	43,63 ± 13,74	37,66 ± 4,65	42,63 ± 10,33
Leydigosomatic index (%)	0,03 ± 0,01	0,04 ± 0,00	0,04 ± 0,00	0,04 ± 0,01	0,04 ± 0,01	0,04 ± 0,01	0,04 ± 0,01

/gt – per gram of testis. Data are expressed as mean ± standard deviation of the mean (n = 6). (*) indicate significant change from control at p ≤ 0.05 (Student Newman Keuls test).

Testosterone concentration

The serum testosterone concentration increased only in animals treated with Chromium VI compared to the control group (Fig. 1).

Histopathology

Data obtained by histopathological analysis are shown in Fig. 2. Slight pathologies, such as vacuoles at the base and apex of the seminiferous epithelium, increased in groups treated with cadmium, lead and nickel (Fig. 2A). Figure 2B shows the pathologies detected.

Cell availability

Animals treated with cadmium and nickel showed initial damage of germ cells and dead cells were observed only in animals treated with cadmium (Fig. 3).

Heavy metals bioaccumulation on testicular tissue

The tested metals were found in normal concentrations in the testes. Analysis of Pb accumulation was not performed due to lack of biological material. Data are shown in Fig. 4.

Oxidative status analyses

SOD activity increased in the group treated with arsenate while GST decreased in groups treated with arsenite, cadmium, lead, chromium VI and nickel, and MDA decreased in all treated groups compared to the control group (Fig. 5).

This is the first study that presents the comparative toxicity of heavy metals arsenate (As ^+ 5), arsenite (As ^+ 3), cadmium (Cd), lead (Pb), chromium VI (Cr VI) and nickel (Ni) in the testes of Swiss mice after acute exposure, via intraperitoneal (Ip) route. Changes in functional, cellular, pathological, and oxidative status parameters were observed.

Biometric, body and testicular data did not change in the treated groups. Such results suggest that the exposure time was not enough to alter these parameters. Similar results were also observed by Lima et al. (2018) [25] with inorganic arsenic, as well as in works carried out with lead acetate [31, 32, 33, 7], chromium VI [24] and nickel [35].

The testicular tissue architecture remained intact after acute exposure of metals, with no notable alterations in the seminiferous tubules such as total length, area and tubule/epithelium ratio. Exposure time and metal concentration can have a great influence on the appearance of changes in these parameters. Rats treated with Cd (50 mg/kg) for 15, 30 and 60 days had decreased tubular diameter (DT) and epithelial height (EH) as treatment increased its duration [36]. Likewise, Chandra et al. (2010) [24] reported that animals exposed to lower concentrations of Cr VI for a longer period showed a decrease in DT. In contrast, Massanyi et al. (2007) [35] observed that animals exposed to higher concentrations of Ni in a shorter time of exposure showed a reduction in DT and EH, in addition to an increase in the lumen diameter (LD). However, similar results to the present study were observed by Lima et al. (2018) [25] in rats after exposure of As⁵⁺ and As³⁺ (0.01 and 10 mg/L for 56 days), by Oliveira (2010) [59] with Ni (0.67 and 1.34 g/L for 60 days) and by Li et al. (2016) [37] and Predes et al. (2016) [38] with Cd (0.2 mg/L for 24 hours and 1.2 mg/kg for 7 and 56 days, respectively).

Regarding the percentage and volume of tubular components, the groups treated with Cd, Pb, Cr VI and Ni showed an increase in the volumetric density of tunica propria which reflected in the increase in its volume, that may be related to initial toxicity of these metals. The relationship between the increase in the tunica propria and spermatogenesis damage is still unknown, but this change can affect the communication between tubular and intertubular compartments [20]. Studies indicate that this increase is a result of the increased production of collagen fibers, probably stimulated by the proteoglycan decorin involved in fibrillogenesis in infertile testes [39, 40, 41]. Studies show that alterations in the proper tunic may be related to oxidative stress [42, 43].

The volumetric density of seminiferous epithelium reduced in the groups exposed to Cd, Pb, Cr VI and Ni with no indication of other apparent alterations. Changes in these parameters were also observed by Neves (2016) [44] with Pb, Saxena et al. (1990) [45] with Cr, Mouro et al. (2019) [7] with Cd and by Toman et al. (2012) [46] with Ni, at higher concentrations and different exposure periods.

The evaluated parameters of cells from the seminiferous epithelium did not undergo significant changes, indicating that the spermatogenic process was not affected, in this way, by the tested metals. Experiment performed by Li et al. (2012) [47], with a similar dose of arsenic, obtained the same results. However, Corpas et al. (2002) [48] and Nava-Hernández et al. (2009) [49] reported that exposure for a longer period to Pb induced significant reductions in cell populations in the seminiferous epithelium. Changes were also reported by Bu et al. (2011) [50] with Cd and by Kong et al. (2014) [51] and Kong et al. (2018) [52] with Ni. At higher doses Cd can induce interstitial edema, hemorrhage and necrosis accompanied by damage to the seminiferous tubules, affecting sperm and their precursors [53, 54]. On the other hand, Cr VI in higher doses than the one used in this study can severely affect meiotic prophase, promoting disturbances in spermatogenesis [55].

The testicular component most sensitive to exposure by external agents is the intertubule [56]. However, the percentage and volume of the intertubule did not change after exposure to heavy metals in this study. Similar results were found by Cupertino et al. (2017) [6] in rats exposed to different concentrations of Cd for 7 days, and by Massanyi et al. (2007) [57] using Pb for 48 hours. The exposure time may have been insufficient for the appearance of alterations, since Souza et al. (2016) [58] reported changes in these parameters in animals exposed to As⁵⁺ and As³⁺ for 56 days. Saxena et al. (1990) [45] also observed changes in these parameters in animals exposed to Cr VI for 90 days. Chronic exposure to Ni can cause a reduction in the volumetric proportion of macrophages accompanied by a reduction in the Leydig cells which may indicate physiological damage to androgenesis [59].

The nuclear diameter and volume of the Leydig cell were reduced in animals exposed to As³⁺ and, on the other hand, as a compensation, the number of Leydig cells in the testes increased in these animals. These alterations represent changes in the intertubular microstructure, indicating a decrease in the size of the Leydig cell which can alter the testosterone production. The changes may have occurred in response to direct poisoning on the cell, through blood vessels or due to the generation of ROS. Similar data were found by Souza et al. (2018) [60], after long exposure, probably due to ROS action. Animals exposed to Pb, Cr VI and Ni showed an increase in the diameter and volume of the Leydig cell nucleus, without other apparent changes. Studies with animals exposed to As⁵⁺, Ni and Pb, for a longer period of time, reported changes in the morphometric parameters of Leydig cells, proving that longer periods of exposure promote greater testicular changes [46, 61, 58].

Testosterone plays a crucial role in the male reproductive system [62]. Testosterone level increased significantly in animals exposed to Cr VI, remaining unchanged in the other groups. According to Castro et al. (2002) [63], there is no correlation between high testosterone concentrations and increased spermatogenic process. The increase in testosterone level in animals exposed to Cr VI did not cause great damage or gains to spermatogenesis. Abarikwu et al. (2018) [36] reported a decrease in testosterone levels only after 15, 30 and 60 days of weekly exposure to Cd (50 mg/kg).

Slight pathologies in the seminiferous tubules, such as vacuoles at the base and apex found in animals exposed to Cd, Pb and Ni are consistent with the initial cell damage found in the groups that received Cd and Ni. Changes in tissue morphology were also found, and these analyses, according to Hinton et al. (1992) [64], are important to demonstrate the toxicity of these metals. Cupertino et al. (2017) [6] found pathologies in the seminiferous tubules of rats intoxicated by Cd (1.4 mg/kg) for 7 days, such as tubular obstruction and vacuolization of germ cells. Predes et al. (2010) [13] and Predes et al. (2016) [38] also reported disorganization of the seminiferous tubules in animals that received 1.2 mg/kg of Cd for 7 days. Additionally, Kong et al. (2014) [51] with Ni and Costa (2013) [61] with Pb reported that animals exposed to these metals also presented pathologies in the seminiferous tubule associated with cellular damage. No pathology was observed in animals exposed to As⁵⁺, As³⁺ and Cr VI. However, studies in animals exposed to these metals for more than 7 days have reported the emergence of pathologies in the seminiferous epithelium [65, 66, 67, 25].

Cell availability analysis indicated initial damage and cell death in the seminiferous tubules of animals treated with Cd. These alterations are associated with slight pathologies and oxidative stress but without alteration in the morphometry of the germinal epithelium. Probably, the exposure time and dose were insufficient to generate more damage, but some morphological and functional alterations started to become apparent. It is known that many metals promote changes that can lead to cell death [68]. In experiments with rats (1 mg/kg Cd for 15 days) and mice (2, 4 and 8 mg/kg Cd for 24 hours), it was possible to observe the imbalance between cell proliferation and apoptosis, thus increasing the apoptotic process [50, 69]. Both the mitochondrial and endoplasmic reticulum pathways are involved in Cd-induced germ cell apoptosis in mice [50, 69].

Initial damage process was observed in the cells of the seminiferous tubule of animals exposed to Ni as well as slight pathologies, but the tubule diameter did not change. Similar data were found by Kong et al, (2014) [51] with rats exposed to Ni at concentrations higher than 1.5 mg/kg. Alarifi et al. (2014) [70] observed that Ni cytotoxicity was concentration and time dependent in epithelial cell culture. Animals exposed to As⁵⁺, As³⁺, Pb and Cr VI did not undergo changes in cell availability, suggesting that the exposure time was a key factor for the onset of changes. Studies with animals exposed to As⁵⁺ and As³⁺ for 56 days [25], Pb for 40 days [71], and Cr VI for 15 consecutive days [67] showed cell damages in the seminiferous tubule as well as slight pathologies.

The statistically non-significant accumulation of the tested metals in the testicles is possibly due to the exposure time, the number of exposures and the form of elimination from the body. Lima et al. (2018) [25] observed an increase in the concentration of As⁵⁺ and As³⁺ in the testicles of rats after 56 days of exposure, a period longer than that used in the present study. Studies indicate that natural gonad defenses against Cd accumulation can be efficient for up to 30 days [72, 13, 36]. These authors suggest that the toxic effects of Cd appear to be limited due to the difficulty in causing damage when bound to the protective protein metallothionein. Animals exposed to Cd (1.5g/L) daily for 3, 6 and 12 months showed increased metal concentration in the testes [56]. On the other hand, removing exposure for 3 and 6 months before euthanasia promoted stability and even decreased metal accumulation in the testis [56]. Although the analysis of lead concentration was not performed in the present study, Adhikari et al. (2001) [73] reported that rats exposed to Pb (5, 10 and 20mg/kg for 2 weeks) showed metal accumulation in the testis. Massanyi et al. (2007) [35] found high concentrations of Ni in the semen of animals exposed daily to the metal, correlating these levels to sperm malformations, indicating that Ni may have managed to invade the organ and cross the defense barrier.

Redox imbalance is the first change that occurs when an organism is intoxicated by heavy metal, generating an imbalance called oxidative stress. To protect the organism from this oxidative damage antioxidant enzymes are produced, such as superoxide dismutase (SOD), which converts superoxide radical (O²⁻) into hydrogen peroxide (H₂O₂) and catalase (CAT) that converts H₂O₂ to H₂O and O₂ [74]. The increase in SOD activity in animals exposed to As⁵⁺ may have occurred due to the increase in O²⁻ production, however without changing the activity of CAT or glutathione-S-transferase (GST). In association, there was a decrease in malondialdehyde (MDA) levels in these same animals, indicating that the first line of defense against harmful effects of heavy metal is efficient. These results agree with the data reported by Lima et al. (2018) [25] where rats exposed to As⁵⁺ (0.01 and 10 mg/L for 56 days) also showed efficiency of the first line of antioxidant defense in the testes.

Animals exposed to As³⁺, Cd, Pb, Cr VI, and Ni showed a reduction in GST activity associated with a decrease in MDA levels. It is understood that SOD and CAT enzymes are acting within the normal range in combating the deleterious effects induced by metals. The reduced activity of GST can be associated with the metal elimination and detoxification. In an attempt to re-establish the balance in the oxidative state, GST may have reached its maximum activity, leading to a decrease in the capacity of H₂O₂ dismutation and in its activity [75]. Another way that also justifies the reduction of GST is the process of metal elimination. The decrease in MDA levels, one of the products of lipid peroxidation, may indicate efficiency of GST activity. It is known that antioxidant enzymes can fluctuate over a period after heavy metal intoxication, as observed with As [76], Cd [36], Pb [10], Cr [77] and Ni [78]. Similar results were observed by Bashandy et al. (2016) [76] showing that exposure to arsenic reduced the level of reduced glutathione (GSH) and CAT activity in rat testes. Depletion of GSH, GST cofactor, can also be attributed to the binding of heavy metals with sulfhydryl groups and subsequent elimination [79]. In general, heavy metal metabolism is known as biotransformation, a process by which the metal becomes more soluble in water and is easily excreted [80, 81, 82].

Thus, we understand that this study's functional, cellular and pathological changes may be an initial response to oxidative stress induced by heavy metal poisoning. Exposure time, concentration, and type of metal are critical factors in the level of induced changes in the male reproductive system. Therefore, after acute exposure to heavy metals (1.5 mg/kg i.p.) for 7 days, Ni was more toxic causing pathological and functional damage in more significant proportions. Cd-induced slight pathologies, initial damage, and cell death in the seminiferous tubule, in addition to functional alterations. Cr VI caused an increase in serum testosterone concentration. Animals intoxicated by Pb have mild pathologies. As3 + promoted changes only in Leydig cells and As5 + did not cause significant changes in the balance of testicular functioning.

Acute exposure to arsenate, arsenite, cadmium, lead, chromium VI, and nickel promotes functional, cellular, pathological, and oxidative stress alterations. The order of toxicity among the tested metals established by comparative damage analysis follows Ni > Cd > Cr VI = Pb > As ^+ 3 > As ^+ 5, In groups with higher toxicity, such as nickel and cadmium were observed functional changes, alterations in the testicular microstructure and cell death. Germ and Leydig cells exhibited divergent resistance profiles for each metal. Exposure time and administered doses are associated with damage caused by heavy metals in the testes. Based on our results, we can point out that the heavy metals tested can be harmful to male reproductive health in low doses and for a short period of exposure. Care must be greater taking into account the damage order defined in this work.

Conflicts of Interest

The authors of this manuscript state that they do not have conflicts of interest to declare.

Data Availability statement

The data supporting the findings of this study are available within the article.

Acknowledgements

The authors are grateful to the support provided by Fundação do Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, processes APQ-01895-16, PPM-00687-17 and PPM-00077-18), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, processes 303972/2017-3, 423594/2018-4, 305093/2017-7 and MCTIC 408503/2018-1), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES, finance code 001).

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Amanda Alves Lozi, Fernanda Carolina Ribeiro Dias, Janaina da Silva, Diane Costa Araujo, Ingred Carol Gonçalves, Fabiana Cristina Silveira Alves de Melo, Elizabeth Lopes de Oliveira, Mariella Bontempo Duca de Freitas and Sérgio Luis Pinto da Matta. The first draft of the manuscript was written by Amanda Alves Lozi and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval

This study was carried out in accordance with ethical principles, approved by the Ethics Committee for Animal Use of the Federal University of Viçosa- UFV - protocol 07/2018.

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Comparative Toxicology of Heavy Metals Arsenate, Arsenite, Cadmium, Chrome Vi, Lead, and Nickel in the Testes of Adult Swiss Mice After Acute Exposure

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Abstract

Figures

Introduction

Materials And Methods

Animals

Chemicals

Experimental design and tissue collection

Biometric parameters

Tubular morphometry

Cell counting

Intertubular morphometry

Testosterone concentration

Histopathology

Cell availability

Heavy metals bioaccumulation on testicular tissue

Oxidative status analyses

Statistical analysis

Results

Biometric parameters

Tubular morphometry

Cell counting

Intertubular morphometry

Testosterone concentration

Histopathology

Cell availability

Heavy metals bioaccumulation on testicular tissue

Oxidative status analyses

Discussion

CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

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