Edited by: Eleonore Fröhlich, Medizinische Universität Graz, Austria
Reviewed by: Jay Manoj Bhatt, The University of Texas at El Paso, United States; Hideko Sone, National Institute for Environmental Studies, Japan
This article was submitted to Predictive Toxicology, a section of the journal Frontiers in Pharmacology
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Mequindox (MEQ), belonging to quinoxaline-di-
As a class of synthetic heterocyclic agents, QdNOs are also known as DNA synthesis inhibitors and are usually used in human and veterinary medicine due to their broad range of biological properties including antibacterial, anti-candida, antitubercular, anticancer, antiprotozoal and growth promoting activities (
The chemical structures of carbadox, olaquindox, and mequindox.
The use of MEQ as an animal feed additives or antimicrobial agents had caused serious health hazard effects. MEQ had adverse effects on pigs and chicken in clinical use (
The literature regarding the reproductive toxicity and teratogenic potential of QdNOs was published previously (
Most current international guidelines require a two generation reproduction test to evaluate the reproductive toxicity of industrial chemicals. Because of no reports of reproductive and development studies of MEQ, it is impossible to determine the NOAEL of developmental or reproductive and evaluate the risk of MEQ. For a long-term perspective, increased use of MEQ without comprehensive safety evaluation is questionable, especially the concerns related to the potential impact on human health of the proposed use in food producing animals. Therefore, the present study was designed to evaluate the two generation reproduction of MEQ in Wistar rats with a wide range of doses according to the following principles (
Mequindox (C11H10N2O3, purity 98%) was obtained from Beijing Zhongnongfa Pharmaceutical Co. Ltd. (Huanggang, China). All other reagents were of analytical grade.
One hundred twenty five male and 125 nulliparous female Wistar rats (
Mequindox was incorporated in diets containing 0, 55, 110, or 275 mg/kg, equivalent to 0, 10, 20, or 50% of the LD50 in diets. A dose of 55 mg/kg diet was selected because it is the normal concentration of OLA permitted in food. Two hundred seventy five milligrams per kilogram dietary level MEQ inhibited feed intake and body weight of the rats (
The two generation feeding reproduction study and teratogenic study were performed according to FDA Toxicological principles for the safety assessment of food ingredients. IV.C.9.a. Guidelines for Reproduction Toxicity Studies (
A graphic representation of the study design is presented in
Experimental design for two generation reproductive and teratogenic test.
For mating, one female and one male were placed into one cage overnight. Successful mating was ascertained by the presence of sperm in the vaginal smear, and the following first 24 h was designated as day 0 of gestation. Mated females were housed singly in clear polycarbonate cages with stainless steel wire lids and were allowed tap water and feed
After 12 weeks pre-breed period, F0 and F1 rats were housed as breeding pairs with the ratio of 1:1, until evidence of mating was observed or for 21 consecutive days. Rats in the F1 generation were bred twice to produce F2a and F2b generations. Copulation was examined every morning and successful mating was confirmed by the presence of vaginal plug. Mated females were housed singly in clear polycarbonate cages with stainless steel wire lids and were allowed tap water and feed
Routine cage-side observations were performed on all animals twice a day throughout the study for general signs of toxicologic and pharmacologic effects, morbidity and mortality, general appearance, and behavior. An expanded set of clinical evaluations, performed outside of the cage, was performed on all animals twice before test material administration and weekly during the study period to detect neurologic disorders, behavioral changes, autonomic dysfunctions, and other signs of nervous system toxicity. Feed intakes of rats were measured weekly. Maternal body weights were measured on GD 0, 7, 14, and 21. Maternal body weight gains were calculated. Throughout gestation, all pregnant rats were observed daily for mortality, morbidity, general appearance and behavior. Females were sacrificed and subjected to external and internal macroscopic examination at the scheduled termination (GD 21). The day of parturition was designated as PND 0. Total litter size and number of live and dead pups were recorded. The live pups were counted, sexed, weighed, and examined grossly on PND 0, 4, 7, 14, and 21.
Each rat was observed at least twice daily throughout the study. Relevant behavioral changes and all signs of toxicity, morbidity, or mortality were recorded. Estrous cycle length and normality were observed daily by vaginal smears for all F0 and F1 females during a minimum of 3 weeks before mating and during cohabitation. The duration of gestation was calculated from day 0 of pregnancy. The mating index [(number of males (or females) with evidence of mating/total number of males (or females) paired) × 100], fertility index [number of females with a confirmed pregnancy/total number of females paired) × 100], live birth rate [(number of females producing live litter/total number of pregnancy) × 100], survival rate of birth [(Survival number of litter on the forth days of birth/total number of surviving litter at birth) × 100], survival rate of lactation (%) [(Survival number of weaned litter on the 21st days of birth/total number of surviving litter at birth) × 100], and sex ratio (expressed as % males per litter) were calculated. Throughout gestation, all pregnant rats were observed daily for mortality, morbidity, general appearance, and behavior. Females were sacrificed and subjected to external and internal macroscopic examination at the scheduled termination (GD 21 or pups weaned).
For the F0 and F1 generation males selected for mating, sperm samples obtained from the right cauda epididymis per testis were collected and then placed in Dulbecco’s phosphate buffered saline (maintained at approximately 37°C) with 10 mg/mL bovine serum ALB. After 10-min incubation, sperm motility was determined under light microscopy (Olympus BX 41, Japan). Sperm (minimum 200 per sample) samples from the cauda epididymis were examined as a fixed wet preparation and classified as either normal (both head and midpiece appear normal) or abnormal (i.e., fusion, isolated heads, misshapen heads, and/or tails).
The dams were euthanized on GD 21. The ovaries and uterus of each rat were removed and the status of all implantation sites, i.e., live and dead fetuses, early and late resorptions, and total implantations were examined. Live fetuses were weighed individually. All live fetuses were sexed, weighed, and inspected for external malformations; including cleft palate. Approximately one-half of the live fetuses in each litter were randomly selected for either skeletal or visceral examination. The skeletal evaluation of 85% ethanol-fixed fetuses was performed after staining the skeleton with Alizarin Red S and clearing with the potassium hydroxide solution. The remaining live fetuses in each litter were fixed in Bouin’s solution prior to dissection. A freehand razor sectioning technique was adapted to detect internal malformations of the head and abdomen.
In each litter, the number of pups, stillbirths, live births, and the presence of gross anomalies were examined after delivery. Dead pups were necropsied and observed for gross defects. The neonates were carefully observed, and their sex and weight were noted on PND 0, 4, 7, 14, and 21.
Serum chemistry was examined using Synchron Clinical System CX4 (Beckman Coulter, Brea, CA, United States) according to the manufacturer’s directions (Beijing Leadman Biochemistry Technology Co. Ltd, Beijing, China). Parameters in serum chemistry included ALB, ALT, AST, TP, CREA, CL-, ALP, UA and so on.
The macroscopical examination of organs/tissues included the visible lesions on the external surface. Each rat received a gross necropsy and microscopic examination of standard tissues including: liver, spleen, kidney, adrenal, testes, epididymis, uterus, and ovary. The organs/tissues were carefully examined and gross lesions were recorded. During necropsies, a complete list of organs and tissues was weighed separately to calculate the organ weights per 100 g body weight (relative organ weight).
All the reproductive organs, including testis, epididymis, prostate, uterus and ovaries were examined macroscopically, and gross lesions were recorded. The tissues from each rat, with the exception of testis, were preserved in 10% neutral-buffered formalin and slides prepared for histopathological examination. Routine paraffin embedding technique was used for histological examination of tissue sections. Sections of 5 μm thickness stained with H&E were examined under light microscopy (Olympus BX 41, Japan) for morphological changes. As a necessary step to determine a no-observed-adverse-effect-level (NOAEL) in target organs, other tissues such as liver and kidneys from treatment groups were also examined histologically.
For pre-breed data, Levene’s test was performed to determine whether the groups had equal variances. If the variances were homogeneous, ANOVA was carried out. If not, they were analyzed by the Kruskal–Wallis non-parametric ANOVA. If either of the tests showed a significant difference among the groups, the data were analyzed by the multiple comparison procedure of the Dunnett’s
Continuous data such as maternal body weight, food consumption, fetal body weight, sperm numbers, body length and tail length were subjected to ANOVA, and Dunnett’s multiple comparison tests were conducted when analytic results were significant. Incidence data such as the gender ratio, external, skeletal and visceral variations, the proportions of litters with malformations and developmental variations were compared using a chi-square test and Fisher’s exact test. Sperm motility and sperm morphology were analyzed with Kruskal-Wallis test with Mann-Whitney
All the F0 male and female rats were survived except three males and one pregnant female were found dead during pre-breeding and mating periods at 275 mg/kg MEQ diet. The rats were weak, emaciated, and showed some neurological signs such as hyperactive, readily irritated and aggressive on handling at the 275 mg/kg MEQ mg/kg group.
The body weights of F0 and F1 male and female rats from pre-bred, mating, gestation and lactation periods are presented in
Mean body weights of F0 female, F0 male, F1 female, and F1 male in the two generation reproductive and teratogenic test.
For males, there is a downward trend in body weight of F0 males at the 25, 55, and 110 mg/kg MEQ groups during most of weeks (except at first and ninth weeks). For F0 males, a significantly reduced body weight was noted at 275 mg/kg MEQ group from weeks 4 to 12. A reduced body weight of F1 males was observed at 55 mg/kg MEQ group from weeks 1 to 3. Body weights of F1 males were significantly decreased from 1 to 12 weeks at 275 mg/kg MEQ group when compared with controls. Decreased body weights from 1 to 5 weeks and 10 to 12 weeks were noted in F1 males at 55 and 110 mg/kg MEQ groups.
Summery of F0 generation mating and F1 litter and survival data (Mean ± SD).
Control | M25 | M55 | M110 | M275 | |
---|---|---|---|---|---|
No of rats (male/female) | 25/25 | 25/25 | 25/25 | 25/25 | 22/25 |
No. of pregnant females | 24 | 23 | 23 | 20 | 19∗ |
Mating index (%) | 96 | 88 | 92 | 80∗∗ | 76∗∗ |
Fertility index (%) | 96 | 88 | 92 | 80∗∗ | 76∗∗ |
Gestation index | 100 | 95.65 | 91.30 | 100 | 94.74 |
Total no. of pups/litter | 9.38 ± 2.19 | 9.4 ± 3.25 | 9.46 ± 2.21 | 8.57 ± 3.88 | 9.57 ± 2.17 |
No. of live pups/litter | 9.38 ± 2.19 | 9.27 ± 3.17 | 9.46 ± 2.21 | 8.57 ± 3.88 | 8.21 ± 3.14∗ |
Sex ratio (%) of F1 pups | 50.7 | 48.2 | 48.1 | 46.6 | 42.5∗ |
Live birth rate (%) | 100 | 98.6 | 100 | 93.3 | 88.1 |
Survival rate of birth (%) | 92.9 | 95.4 | 82.2 | 83.8 | 56.6∗∗ |
Survival rate of lactation (%) | 98.0 | 99.1 | 94.6 | 89.4 | 93.4 |
No. of total litter losses | 0 | 0 | 1 | 2 | 4∗ |
Mean pup body weights day 1 (g) | 6.30 ± 0.95 | 6.33 ± 0.57 | 5.91 ± 0.71 | 6.26 ± 0.66 | 5.40 ± 0.69 |
Mean pup body weights day 4 (g) | 12.47 ± 1.65 | 11.9 ± 1.67 | 10.09 ± 1.13 | 10.06 ± 2.02 | 8.11 ± 1.63 |
Mean pup body weights day 7 (g) | 17.0 ± 1.7 | 16.9 ± 1.5 | 15.9 ± 2.1 | 15.3 ± 1.6 | 14.12 ± 2.5 |
Mean pup body weights day 14 (g) | 23.51 ± 3.09 | 24.6 ± 4.32 | 23.19 ± 3.64 | 20.95 ± 2.92 | 19.40 ± 2.54 |
Mean pup body weights day 21 (g) | 36.87 ± 5.30 | 39.2 ± 7.55 | 34.35 ± 4.43 | 32.29 ± 4.84 | 22.42 ± 4.28 |
The reproductive parameters of F1 parent/F2 offspring were shown in
Summery of F1 generation mating and F2 litter and survival (Mean ± SD).
Control | M25 | M55 | M110 | M275 | |
---|---|---|---|---|---|
No of rats (male/female) | 25/25 | 25/25 | 25/25 | 25/25 | 25/25 |
No. of pregnant females | 23 | 20 | 21 | 18 | 14∗ |
Mating index (%) | 91.7 | 83.3 | 87.5 | 75∗∗ | 58.3∗∗ |
Fertility index (%) | 91.7 | 83.3 | 87.5 | 75∗∗ | 58.3∗∗ |
Gestation index | 95.46 | 100 | 95.24 | 100 | 100 |
Total no. of pups/litter | 10.17 ± 2.44 | 8.94 ± 2.18 | 9.0 ± 2.69 | 9.12 ± 3.04 | 7.46 ± 1.90∗ |
No. of live pups/litter | 10.17 ± 2.44 | 8.67 ± 2.91 | 8.53 ± 2.92 | 8.35 ± 2.89 | 7.38 ± 1.89∗ |
Sex ratio (%) of F2 pups | 50.82 | 58.39∗ | 47.92 | 51.41 | 45.36 |
Live birth rate (%) | 99.8 | 90.5 | 87.87∗ | 93.1 | 78.8∗∗ |
Survival rate of birth (%) | 97 | 97.9 | 79.3∗∗ | 85.2∗ | 78.0∗∗ |
Survival rate of lactation (%) | 100 | 82.9 | 69.6∗ | 78.2∗∗ | 93.8 |
No. of total litter losses | 0 | 1 | 2 | 3 | 3∗ |
Mean pup body weights day 1 (g) | 5.99 ± 0.83 | 6.17 ± 0.88 | 6.19 ± 0.68 | 5.93 ± 0.82 | 5.62 ± 1.03 |
Mean pup body weights day 4 (g) | 13.15 ± 2.30 | 11.14 ± 0.94 | 12.4 ± 2.07 | 11.89 ± 1.44 | 13.03 ± 0.98 |
Mean pup body weights day 7 (g) | 17.1 ± 1.91 | 17.4 ± 1.82 | 17.0 ± 1.7 | 15.5 ± 1.22 | 15.1 ± 1.71 |
Mean pup body weights day 14 (g) | 22.1 ± 2.98 | 19.0 ± 3.21 | 20.9 ± 3.4 | 21.72 ± 3.22 | 20.02 ± 1.73 |
Mean pup body weights day 21 (g) | 31.38 ± 6.05 | 30.52 ± 7.2 | 32.02 ± 5.44 | 28.2 ± 7.03 | 30.47 ± 2.56 |
The results of serum clinical chemistry of F0 and F1 parents were presented in
Serum clinical chemistry parameters of F0 and F1 Wistar rats.
Parameters | Control | M25 | M55 | M110 | M275 |
---|---|---|---|---|---|
ALT (IU/L) | 59.11 ± 11.03a | 76.31 ± 30.52 | 67.76 ± 11.10 | 78.5 ± 35.25 | 79.41 ± 18.51 |
AST (IU/L) | 121.0 ± 21.83 | 121.6 ± 25.51 | 150.0 ± 30.80 | 141.6 ± 27.31 | 243.7 ± 61.67∗ |
ALP (IU/L) | 35.71 ± 7.03 | 46.10 ± 14.85 | 35.51 ± 7.92 | 48.1 ± 15.58 | 49.82 ± 8.61 |
TP (g/L) | 71.20 ± 2.96 | 73.32 ± 5.28 | 73.22 ± 5.26 | 75.31 ± 5.62 | 82.13 ± 8.48 |
ALB (g/L) | 34.50 ± 3.14 | 31.31 ± 1.64 | 35.41 ± 3.29 | 33.32 ± 1.45 | 33.81 ± 1.74 |
CREA (mmol/L) | 48.90 ± 7.32 | 53.60 ± 7.65 | 50.01 ± 9.72 | 55.51 ± 9.56 | 59.75 ± 9.34∗ |
BUN (mmol/L) | 8.9 ± 0.88 | 7.6 ± 1.29 | 7.7 ± 0.41 | 7.8 ± 1.21 | 7.7 ± 0.23 |
UA (μmol/L) | 132.5 ± 27.63 | 113.8 ± 9.61 | 126.3 ± 14.44 | 115.8 ± 11.56 | 105.6 ± 16.6∗ |
Na (mmol/L) | 126.2 ± 2.84 | 124.0 ± 2.38 | 131.7 ± 3.68∗∗ | 126.0 ± 2.83 | 120.7 ± 2.63∗∗ |
K (mmol/L) | 4.3 ± 0.63 | 4.9 ± 1.09 | 5.4 ± 0.94 | 5.9 ± 0.89 | 6.4 ± 0.86∗∗ |
CL (mmol/L) | 104.0 ± 3.08 | 101.9 ± 2.25 | 105.6 ± 4.5 | 104.9 ± 2.52 | 105.7 ± 8.9 |
ALT (IU/L) | 78.50 ± 9.07 | 87.42 ± 12.34 | 80.31 ± 14.56 | 89.5 ± 12.97 | 132.9 ± 28.61∗ |
AST (IU/L) | 173.0 ± 30.07 | 177.5 ± 46.1 | 171.7 ± 48.71 | 179.7 ± 46.6 | 231.3 ± 43.74∗ |
ALP (IU/L) | 81.71 ± 11.08 | 73.01 ± 14.23 | 73.92 ± 10.91 | 76.0 ± 14.01 | 73.41 ± 16.24 |
TP (g/L) | 75.61 ± 2.75 | 79.13 ± 2.15 | 80.9 ± 2.08 | 81.22 ± 4.06 | 91.24 ± 9.41∗ |
ALB (g/L) | 33.30 ± 3.83 | 34.53 ± 2.34 | 34.4 ± 1.61 | 36.72 ± 1.59 | 37.72 ± 1.19 |
CREA (mmol/L) | 50.30 ± 4.7 | 57.42 ± 5.32 | 54.4 ± 6.43 | 59.61 ± 5.71 | 67.61 ± 8.13 |
BUN (mmol/L) | 8.6 ± 1.07 | 7.3 ± 0.12 | 8.2 ± 0.55 | 7.5 ± 0.98 | 7.6 ± 1.23 |
UA (μmol/L) | 94.70 ± 15.71 | 90.42 ± 16.54 | 91.81 ± 31.18 | 91.91 ± 16.23 | 90.31 ± 20.04 |
Na (mmol/L) | 158.5 ± 3.64 | 151.3 ± 4.38 | 151.5 ± 2.65 | 153.5 ± 4.88 | 141.8 ± 2.62∗∗ |
K (mmol/L) | 6.6 ± 0.94 | 7.1 ± 3.25 | 6.9 ± 1.61 | 7.5 ± 3.14 | 9.7 ± 2.38∗∗ |
CL (mmol/L) | 101.8 ± 8.87 | 105.6 ± 1.82 | 102.8 ± 4.44 | 107.7 ± 1.19 | 104.9 ± 2.94 |
At sacrifice, the body weights and organ weights of F0 and F1 females were weighed. At 275 mg/kg MEQ group, significant changes of organ weights of liver, spleen, adrenal, ovary, and uterus were found in F0 females (
Organ weight (g) of F0 female Wistar rats.
Organs | Control | M25 | M55 | M110 | M275 |
---|---|---|---|---|---|
Final body | 268.0 ± 20.6 | 270.4 ± 47.58 | 266.6 ± 38.52 | 261.0 ± 37.46 | 240.7 ± 33.53∗ |
Absolute (g) | |||||
Brain | 1.34 ± 0.09 | 1.34 ± 0.08 | 1.34 ± 0.08 | 1.31 ± 0.08 | 1.29 ± 0.09 |
Liver | 5.83 ± 0.45a | 7.11 ± 1.43∗ | 6.54 ± 1.42 | 6.75 ± 1.73 | 7.85 ± 1.47∗ |
Spleen | 0.48 ± 0.10 | 0.51 ± 0.1 | 0.51 ± 0.12 | 0.52 ± 0.1 | 0.68 ± 0.12∗ |
Kidneys | 0.76 ± 0.06 | 0.84 ± 0.16 | 0.73 ± 0.11 | 0.69 ± 0.15 | 0.81 ± 0.13 |
Adrenal | 0.046 ± 0.011 | 0.041 ± 0.007 | 0.038 ± 0.007 | 0.033 ± 0.009∗ | 0.025 ± 0.010∗ |
Uterus | 3.12 ± 1.97 | 3.74 ± 1.68 | 3.90 ± 1.83 | 3.73 ± 1.89 | 2.35 ± 1.73∗ |
Ovary | 0.38 ± 0.16 | 0.29 ± 0.17 | 0.31 ± 0.26 | 0.26 ± 0.13 | 0.11 ± 0.32∗ |
Brain | 0.44 ± 0.04 | 0.45 ± 0.04 | 0.48 ± 0.04∗ | 0.55 ± 0.04∗∗ | 0.56 ± 0.05∗∗ |
Liver | 2.50 ± 0.18 | 2.67 ± 0.35 | 2.72 ± 0.41 | 3.12 ± 0.68∗ | 3.23 ± 0.34∗ |
Spleen | 0.21 ± 0.03 | 0.20 ± 0.03 | 0.21 ± 0.03 | 0.21 ± 0.03∗ | 0.28 ± 0.04∗ |
Kidneys | 0.33 ± 0.04 | 0.31 ± 0.04 | 0.31 ± 0.03 | 0.32 ± 0.05 | 0.33 ± 0.03 |
Adrenal | 0.019 ± 0.007 | 0.016 ± 0.005 | 0.016 ± 0.004 | 0.015 ± 0.003 | 0.009 ± 0.004∗ |
Uterus | 1.26 ± 0.23 | 1.19 ± 0.32 | 1.06 ± 0.34 | 1.23 ± 0.32 | 1.72 ± 0.25∗∗ |
Ovary | 0.07 ± 0.09 | 0.07 ± 0.02 | 0.06 ± 0.01 | 0.08 ± 0.04 | 0.05 ± 0.05∗ |
Organ weight (g) of F1 female Wistar rats.
Organs | Control | M25 | M55 | M110 | M275 |
---|---|---|---|---|---|
Final body | 301.8 ± 33.2 | 298.6 ± 46.3 | 264.6 ± 36.9∗∗ | 268.0 ± 42.5∗∗ | 205.1 ± 27.9∗∗ |
Absolute (g) | |||||
Brain | 1.49 ± 0.07 | 1.52 ± 0.05 | 1.50 ± 0.07 | 1.46 ± 0.11 | 1.48 ± 0.07 |
Liver | 8.87 ± 1.95a | 8.40 ± 2.18 | 7.17 ± 1.88∗ | 8.31 ± 2.33 | 7.10 ± 1.90∗∗ |
Spleen | 0.64 ± 0.22 | 0.56 ± 0.12 | 0.54 ± 0.13 | 0.55 ± 0.17 | 0.54 ± 0.11 |
Kidneys | 0.81 ± 0.24 | 0.82 ± 0.21 | 0.78 ± 0.23∗ | 0.76 ± 0.23∗ | 0.65 ± 0.16∗∗ |
Adrenal | 0.047 ± 0.033 | 0.040 ± 0.015 | 0.050 ± 0.060 | 0.065 ± 0.122 | 0.028 ± 0.008 |
Ovary | 0.22 ± 0.11 | 0.21 ± 0.09 | 0.15 ± 0.05∗ | 0.19 ± 0.10 | 0.13 ± 0.04∗∗ |
Brain | 0.29 ± 0.02 | 0.30 ± 0.02 | 0.30 ± 0.03 | 0.34 ± 0.03∗∗ | 0.36 ± 0.03∗∗ |
Liver | 2.92 ± 0.40 | 2.79 ± 0.39 | 2.72 ± 0.39 | 3.07 ± 0.51∗ | 3.45 ± 0.72∗∗ |
Spleen | 0.21 ± 0.07 | 0.19 ± 0.03 | 0.21 ± 0.05 | 0.21 ± 0.05 | 0.27 ± 0.05∗∗ |
Kidneys | 0.30 ± 0.07 | 0.28 ± 0.09∗ | 0.29 ± 0.08 | 0.28 ± 0.07 | 0.32 ± 0.09 |
Adrenal | 0.015 ± 0.010 | 0.013 ± 0.004 | 0.021 ± 0.030 | 0.023 ± 0.037 | 0.014 ± 0.005 |
Ovary | 0.037 ± 0.016 | 0.036 ± 0.017 | 0.028 ± 0.008 | 0.034 ± 0.015 | 0.032 ± 0.010∗ |
For males, significant decrease in organ weights of brain, kidneys, adrenal, testis, and epididymis were observed in 275 mg/kg MEQ F0 male group (
Organ weight (g) of F0 male Wistar rats.
Organs | Control | M25 | M55 | M110 | M275 |
---|---|---|---|---|---|
Final body | 390.53 ± 36.17 | 391.37 ± 33.32 | 404.57 ± 42.85 | 402.12 ± 65.46 | 335.64 ± 40.57∗∗ |
Absolute (g) | |||||
Brain | 1.77 ± 0.19a | 1.88 ± 0.16 | 1.78 ± 0.25 | 1.48 ± 0.14∗∗ | 1.43 ± 0.12∗∗ |
Liver | 10.1 ± 1.27 | 9.94 ± 1.43 | 10.98 ± 1.89 | 11.15 ± 2.37 | 9.63 ± 1.26 |
Spleen | 0.77 ± 0.17 | 0.76 ± 0.16 | 0.85 ± 0.37 | 0.75 ± 0.10 | 0.71 ± 0.16 |
Kidneys | 1.23 ± 0.19 | 1.22 ± 0.09 | 1.24 ± 0.13 | 1.20 ± 0.23 | 1.03 ± 0.14∗ |
Adrenal | 0.048 ± 0.020 | 0.043 ± 0.016 | 0.034 ± 0.006∗ | 0.032 ± 0.009∗∗ | 0.030 ± 0.005∗∗ |
Testis | 3.46 ± 0.33 | 3.36 ± 0.30 | 3.20 ± 0.42 | 3.30 ± 0.27 | 2.79 ± 0.37∗ |
Epidedmius | 1.37 ± 0.31 | 1.35 ± 0.21 | 1.36 ± 0.23 | 1.30 ± 0.22 | 1.12 ± 0.32∗ |
Brain | 0.35 ± 0.03 | 0.37 ± 0.03 | 0.36 ± 0.03 | 0.34 ± 0.02 | 0.35 ± 0.03 |
Liver | 2.58 ± 0.25 | 2.53 ± 0.23 | 2.71 ± 0.38 | 2.77 ± 0.32 | 2.87 ± 0.38∗ |
Spleen | 0.20 ± 0.05 | 0.19 ± 0.04 | 0.21 ± 0.09 | 0.19 ± 0.03 | 0.21 ± 0.04 |
Kidneys | 0.32 ± 0.04 | 0.31 ± 0.02 | 0.31 ± 0.04 | 0.30 ± 0.02 | 0.31 ± 0.03 |
Adrenal | 0.012 ± 0.005 | 0.011 ± 0.004 | 0.009 ± 0.001 | 0.008 ± 0.002 | 0.009 ± 0.002 |
Testis | 0.87 ± 0.021 | 0.87 ± 0.017 | 0.88 ± 0.036 | 0.87 ± 0.025 | 1.02 ± 0.036∗∗ |
Epidedmius | 0.35 ± 0.06 | 0.30 ± 0.06 | 0.34 ± 0.05 | 0.33 ± 0.06 | 0.33 ± 0.08 |
Organ weight (g) of F1 male Wistar rats.
Organs | Control | M25 | M55 | M110 | M275 |
---|---|---|---|---|---|
Final body | 393.5 ± 30.4 | 390.5 ± 29.0 | 362.1 ± 32.8∗ | 401.4 ± 47.2 | 279.7 ± 41.8∗ |
Absolute (g) | |||||
Brain | 1.92 ± 0.16a | 1.92 ± 0.12 | 1.90 ± 0.15 | 1.64 ± 0.10∗∗ | 1.57 ± 0.13∗∗ |
Liver | 12.4 ± 1.16 | 12.3 ± 1.46 | 12.07 ± 1.47∗ | 13.06 ± 1.92 | 10.17 ± 1.99∗ |
Spleen | 0.69 ± 0.10 | 0.66 ± 0.11 | 0.62 ± 0.10 | 0.66 ± 0.08 | 0.52 ± 0.10 |
Kidneys | 2.31 ± 0.26 | 2.38 ± 0.31 | 2.26 ± 0.30 | 2.20 ± 0.24 | 1.67 ± 0.29∗ |
Adrenal | 0.056 ± 0.022 | 0.056 ± 0.010 | 0.051 ± 0.010 | 0.068 ± 0.023∗ | 0.046 ± 0.008 |
Testis | 3.38 ± 0.36 | 3.41 ± 0.41 | 3.65 ± 0.39 | 3.64 ± 0.79 | 3.57 ± 1.07 |
Epidedmius | 1.10 ± 0.21 | 1.37 ± 0.64 | 1.04 ± 0.32 | 1.05 ± 0.13 | 0.79 ± 0.13∗ |
Brain | 0.38 ± 0.02 | 0.37 ± 0.02 | 0.38 ± 0.01 | 0.38 ± 0.02 | 0.38 ± 0.02 |
Liver | 3.21 ± 0.29 | 3.19 ± 0.18 | 3.36 ± 0.29 | 3.27 ± 0.22 | 3.64 ± 0.38∗ |
Spleen | 0.18 ± 0.02 | 0.17 ± 0.03 | 0.17 ± 0.02 | 0.17 ± 0.02 | 0.19 ± 0.03 |
Kidneys | 0.60 ± 0.04 | 0.62 ± 0.08 | 0.63 ± 0.07 | 0.55 ± 0.05 | 0.60 ± 0.03 |
Adrenal | 0.015 ± 0.006 | 0.015 ± 0.003 | 0.014 ± 0.002 | 0.017 ± 0.006 | 0.017 ± 0.004 |
Testis | 0.87 ± 0.09 | 0.87 ± 0.11 | 0.91 ± 0.056 | 0.91 ± 0.13 | 1.05 ± 0.25∗ |
Epidedmius | 0.29 ± 0.07 | 0.36 ± 0.18 | 0.29 ± 0.09 | 0.28 ± 0.03 | 0.27 ± 0.05 |
As shown in
Selected microphotographs of liver, kidney, adrenal gland, testis and uterus (200X and 400X). M275, 275 mg/kg diet.
In present study, first time two-generation reproductive toxicity study was performed to further evaluate the potential effects of MEQ on reproduction of rats, which provided the information about adverse effects of MEQ on reproduction of rats.
In the two generation reproduction test, a significant decrease in the body weight from fifth week and a significant decrease in feed consumption in most days of week were observed at 275 mg/kg MEQ F0 female group. These results suggested that MEQ caused toxicity to the body weight and food consumption of rats. This finding was similar to a sub-chronic feeding test of MEQ, which demonstrated that the decreased feed consumption was accounted for the decrease in body weight (
In current study, a significant loss of body weight in females were found at 110 and 275 mg/kg MEQ groups, which indicated the maternal toxicity caused by MEQ. MEQ produced significant lower sex ratio in the live pups, viability indices and number of litter loss at 275 mg/kg MEQ diet as compared to the control. The indices of mating and fertility were significantly decreased in 110 and 275 mg/kg MEQ groups. There is a downward trend in the body weight of F1 pups on days 1, 4, 7, 14, and 21 at 55, 110, and 275 mg/kg MEQ groups, and F2 pups on days 1, 4, 7, 14, and 21 at 110 and 275 mg/kg MEQ groups when compared with control group. Our findings suggested that exposure of MEQ at 110 and 275 mg/kg diets induced the developmental inhibition on pups in both generations, while the 55 mg/kg diet caused toxicity only in F1 generations. It was reported that higher dose of cyadox and quinocetone resulted in the significant decrease in the body weight of pups (
The QdNOs derivatives, such as CBX and OLA, were reported to have reproductive and teratogenic toxicity. An aqueous solution of CBX was administered by oral gavage once daily to groups of 10 pregnant rats at a dose of 0, 10, 25, 50, and 100 mg/kg b.w. (
Liver was identified as one of the main target organs for toxicity mediated by MEQ in mice (
The adrenal is considered as a common toxicological target for QdNOs in the endocrine system (
The previous studies reported that the DNA damage is firmly linked to teratogenicity (
In conclusion, the results of two generation feeding reproduction study which we described here provide a more comprehensive toxicity profile of MEQ and benefit evaluating the further risk of MEQ in food animals. MEQ induced maternal, embryo, and reproductive toxicities in rats at the doses of 110 and 275 mg/kg. MEQ depressed the development of fetus and induced teratogenicity at 55, 110, and 275 mg/kg diets. The NOAEL for reproduction toxicity of MEQ was 25 mg/kg diet. Furthermore, the present study indicted that the severe maternal toxicity of MEQ might be responsible for the adverse effects on the maternal rats, conceptus and embryo, which finally, resulting in fetal malformations and fetal deaths.
ZY, XW, and SX conceived the idea. QL and ZL analyzed and discussed the data and wrote the paper. QW, IA, MS, and ZF performed and revised the experiments. All the authors discussed the results and contributed to the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
albumin
alkaline phosphatase
alanine aminotransferase
analysis of variance
aspartate aminotransferase
body weight
blood urea nitrogen
carbadox
chloride
creatinine
deoxyribonucleic acid
gestation day
hematoxylin and eosin
potassium
mequindox
sodium
no observed adverse effect level
olaquindox
postnatal day
quinoxaline-di-
total protein
uric acid