Maternal and developmental toxicity of manganese in the mouse

Maternal and developmental toxicity of manganese in the mouse

45 Toxicology Letters, 69 (1993) 45-52 Q 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4274/93/$06.00 TOXLET 02920 Maternal and de...

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45

Toxicology Letters, 69 (1993) 45-52 Q 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4274/93/$06.00

TOXLET 02920

Maternal and developmental toxicity of manganese in the mouse

Domenec J. SBnchez”, JosC L. Domingoa, Juan M. Llobet” and Carl L. Keenb “Laboratory

of Toxicology and Biochemistry, School of Medicine, ‘Rovira i Virgil? University, Reus (Spain)

and bDepartment of Nutrition, University of California, Davis, CA (USA)

(Received 30 October 1992) (Revision received 8 January 1993) (Accepted 11 January 1993) Key words: Manganese (II) chloride; Mice; Maternal Toxicity; Embryolethality;

Fetotoxicity

SUMMARY Manganese (II) chloride tetrahydrate was investigated in Swiss mice for maternal and developmental toxicity after subcutaneous (s.c.) exposure to doses of 0,2,4, 8 and 16 mg/kg per day from gestation day 6 through 15. Females were sacrificed on gestation day 18, and fetuses were examined for external, visceral, and skeletal abnormalities. Maternal toxicity included significant reductions in weight gain and food consumption at 8 and 16 mg/kg/day, as well as several treatment-related deaths in the high dose-group. There were no treatment-related effects on the number of tota implants, early resorptions, dead fetuses or sex ratio, whereas a significant increase in the number of late msorptions was found in the 4, 8, and 16 mg/kg/day groups. Fetotoxicity, consisting primarily of reduced fetal body weight and an increased incidence of morphological defects was also observed at 8 and 16 mg/kg/day. There were no differences between control and manganese-treated groups in the incidence of individual or total malformations. The no observable adverse effect level (NOAEL) for maternal toxicity of MnCl,.4H,O in mice was 4 mg/kg/day, while the NOAEL for embryo/fetal toxicity was 2 mg/kg/day.

INTRODUCTION

Manganese (MIX]is considered to be an essential trace element for all living mammals. Dietary M.n deficiency can resuit in a wide variety of structural and metabolic defects. A relationship between Mn and carbohydrate metabolism is now well recog-

Correspondence to: Dr. Jose L. Domingo, Laboratory of Toxicology and Biochemistry, School of Medicine, San Lorenzo 21,432Ol Reus, Spain.

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nized, while Mn is also a cofactor for a number of enzymatic reactions, particularly those involved in phosphorylation, cholesterol, and fatty acid synthesis [l-3]. Some CNS disorders has also been associated with Mn deficiency [4]. On the other hand, Mn toxicity represents a serious health hazard in humans. Toxic intakes of the element (either through the air or diet) may result in severe pathologies particularly of the CNS [5-71. However, the neurotoxic effects of chronic and continous Mn exposure on the developing nervous system have not been extensively investigated [8,9]. In addition to extensive neural damage, reproductive and immune system dysf~ction, nephritis, testicular damage, pancreatitis, and hepatic damage can occur with Mn toxicity, but the frequency of these disorders is not known Ph71. Historically, severe exposure to Mn has been restricted mainly to adult male workers in Mn-based industries [IO-131. Recent studies showed that exposure of workers to airborne Mn at low levels such as 0.19-1.39 mg/m” [12], 0.948 mg!m” 1131,or 1 mg/m3 for less than 20 years [lo] may present preclinical signs of Mn intoxication, and disturbances of the same type as parkinsonism. These results seem to indicate that the present exposure standards for Mn (5 mg/m’ in most countries) are not sufficient to protect workers to negative effects. Moreover, reduced fertility in male workers who were moderately exposed to Mn dust in a factory producing Mn salts and oxides (0.07-8.61 mg,/m’; median: 0.97 mg/m3) has also been reported 1141. The possibility that excess exposure to Mn may occur in the vicinity of some refineries or even in the urban general environment, in addition to classical occupational exposure in Mn refineries or auto-repair workshops should also be taken into account [15-l 71. Consequently, Mn toxicity may be a serious health hazard for women and children living in high-exposure environments [18]. In previous studies, teratogenicity was not observed in hamsters [19], rats [20,21], or mice [22] following parenteral or oral Mn exposure during pregnancy. However, embryolethality was found in hamsters when given an i.p. injection of Mn on day 8 of gestation. In order to extend the knowledge of the developmental toxicity of Mn, the present study was undertaken to evaluate the embryotoxic and fetotoxic (including teratogenic) potential of Mn administered S.C.during organogenesis to mice. MATERIALS AND METHODS

Animals and chemicals

Sexually mature (28-32 g) male and female Swiss albino mice were obtained from Interfauna Iberica (Barcelona, Spain), Following an acclimation period of 1 week, two females were housed overnight with one male and checked in the morning for the presence of a vaginal plug, denoted as gestation day (gd) 0. The plug-positive females were randomly distributed into five groups, with 20 animals in each group, and were held under controlled conditions of temperature (22 + 2”C), humidity (50 If: IO%), and lighting (12: 12 h li~~dark cycle). All animals were offered Panlab rodent chow (Panlab, Barcelona) and water ad libitum. Manganese chloride tetrahydrate

(MnCl,*4H,O), analytical grade was obtained from E. Merck (Darmstadt, Germany). Solutions of MnCl, were prepared fresh daily in 0.9% saline and the concentrations adjusted so that a 25-g mouse would receive 0.10 ml. Treatment

Pregnant mice were treated daily with solutions of manganese chloride tetrahydrate at concentrations of 0.50, 1.O, 2.0, and 4.0 mg/ml, given S.C.to obtain dosages of 2,4, 8, and 16 mg/kg/day of MnCl,*4H,O on days 6-15 of gestation. No reactions at the injection site were observed. In a preliminary experiment, the S.C. LD,, of MnCl,e4H,O was found to be 320 mg/kg (single dose). Control animals received similar volumes of 0.9% saline. Body weight, food consumption, and general appearance were monitored daily. Dams were killed with ether on gd 18. The maternal liver and kidney, and gravid uterus were removed and weighed. Uterine contents (i.e., number of implantations sites, resorptions, dead fetuses, and live fetuses) were monitored. All live fetuses were dissected from the uterus and evaluated for body weight and external abnormalities. Approximately one-third of the fetuses were fixed in Bouin’s solution to study visceral anomalies [23]. The remaining fetuses were fixed in 95% ethanol, cleared, and stained with Alizarin red S and examined for skeletal defects [24]. Data analysis

The unit of comparison was the pregnant female or the litter. Kruskal-Wallis analysis of variance procedures were employed to assess the overall effects of MnCl,. Pairwise comparisons were made by the Mann-Whitney U-test. Statistically significant differences between control and test groups were analyzed by a two-tailed Student’s t-test. Significance levels were chosen at P < 0.05. RESULTS

Treatment-related mortality was observed only in the high-dose group, as 32% (6/19) of the dams died prior to scheduled necropsy. Statistically significant reductions in both the maternal body weight and food consumption were observed at 8 mg/kg in the post-treatment period (gd 15-l 8) and at 16 mg/kg in the treatment (gd 6-l 5) and post-treatment (gd 15-18) periods (data not shown). There was a significant decrease in corrected body weight (body weight at termination minus gravid uterine weight) at 8 and 16 mg/kg/day relative to the value in controls (Table I). Gravid uterine weights were significantly decreased in the 8 and 16 mg/kg/day groups, although the corrected body weight change was unaffected by Mn treatment. Relative, but not absolute, maternal liver weights were decreased significantly below controls at 16 mg/kg/day, whereas absolute and relative kidney weights were increased above controls at this dose (Table I). Evaluation of gestational parameters (Table II) indicated no significant treatmentrelated effects on the number of total implants, early resorptions, dead fetuses, or sex

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ratio. A significant increase in the number of late resorptions was found in the 4, 8, and 16 mg/kg/day groups. In addition, there were seven litters with 100% resorptions

TABLE I BODY AND ORGAN WEIGHTS AT TERMINATION NESE CHLORIDE TETRAHYDRATE

FOR MICE TREATED WITH MANGA-

Dose (mglkg per day) 2

0 No. of dams 19 Body weight (g) 61.2 ?r 4.1 Gravid uterine weight (g) 20.7 f 1.8 Corrected body weight (g) 40.5 _+3.2 Corrected body weight change (g)” 8.5 f 2.2 Liver weight (g) 3.1 f 0.4 Relative liver weight (%) 1.7 + 1.3 Kidney weight (g) 0.48 f 0.05 Relative kidney weight (%y 1.18 * 0.12

17 60.7 f 19.6 f 41.1 + 7.0 f 3.2 + 7.8 f 0.48 f 1.17 +

8.0 5.3 3.9 2.1 0.6 1.0 0.04 0.08

4

8

16

17 59.3 + 8.8 18.3 f 7.8 41.0 _+2.7 6.8 f 2.2 3.0 f 0.4 7.3 f 0.9 0.50 + 0.05 1.21 f 0.07

18 47.2 f 6.0’ 9.9 f 6.0r 37.3 f 3.6d 6.7 It 2.8 2.7 f 0.3 7.2 + 1.0 0.45 f 0.04 1.21 f 0.05

13 44.8 f 3.4 f 41.4 f 8.5 + 2.9 f 7.0 f 0.53 + 1.28 +

4.4’ 1.4’ 3.6 2.5 0.2 0.3d 0.07d 0.13d

Values indicate mean f SD. a Corrected body weight = body weight at termination - gravid uterine weight. bCorrected body weight change = corrected body weight - body weight on gestational day 0. ’ Calculated as percentage of corrected body weight, d.e,FSignificantly different from controls (P < 0.05; P < 0.01; P < 0.001, respectively).

TABLE II GESTATIONAL PARAMETERS AND FETAL WEIGHTS IN MICE FETUSES FOLLOWING MATERNAL EXPOSURE TO MANGANESE CHLORIDE TETRAHYDRATE Dose (mg/kg per day)

No. of dams No. of total implants/litter No. of live fetuses/litter No. of non-viable implants/litter Early resorptions Late resorptions Dead fetuses Sex ratio (m/f) Average fetal body weight/litter (g)

0

2

4

8

16

19 13.6 + 2.5 12.7 f 3.3

17 13.1 f 3.0 10.6 f 3.0

17

12.5 f 5.1 10.0 + 4.7

18 11.7 f 4.6 4.2 f 4.0b

13 14.0 f 1.6 0.3 f 0.6b

0.7 f 0.1 * 0.0 + 1.17 + 1.18 f

1.2 f 0.7 + 0.6 + 1.24 f 1.17 f

0.6 + 1.4 f 0.5 + 1.07 + 1.13 f

1.6 f 4.1 f I.7 f 1.01 f 0.97 f

0.7 0.3 0.0 0.96 0.13

1.0 1.1 0.9 0.62 0.09

Values indicate mean + SD. a.b Significantly different from controls (P < 0.01; P < 0.001, respectively).

0.6 0.8” 0.7 0.72 0.12

2.0 1.9 + 3.1 2.8b 11.6 + 4.1b 2.7 0.2 f 0.3 0.50 1.13 + 0.81 0.1 lb 0.82 f O.lOb

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at 16 mg/kg/day. The average fetal body weight per litter decreased as MnCI, dose was increased, and was significantiy below controls at 8 and 16 mg/kg/day. The type and frequency of morphological defects observed in this study are presented in Table III. There was no significant increase in the number of litters with one or more affected fetuses in any treatment group relative to the control for individual or for total external (data not shown) and visceral anomalies, whereas the incidences of wavy ribs and delayed or reduced ossifications in the sternebra, parietal, and occipital were statistically significant (P C 0.05).

TABLE III MORPHOLOGICAL DEFECTS IN MICE FETUSES FOLLOWING MATERNAL EXPOSURE TO MANGANESE CHLORIDE TETRAHYDRATE Dose (mg/kg per day) 0

No. fetuses examined viscerally (No. litters) 103 (19) Enlarged heart Fetuses affected 0 Litters affected 0 Renal hypoplasia Fetuses affected 0 Litters affected 0 No. fetuses examined skeletally (No. litters) 139 (19) Assymetrical sternebrae Fetuses affected 16 Litters affected 9 Wavy ribs Fetuses affected 0 Litters atfected 0 Dorsal hyperkiphosis Fetuses affected 0 Litters affected 0 Steruebrae, delayed ossiiication Fetuses affected 0 Litters affected 0 Parietal bone, reduced ossification Fetuses affected 0 Litters affected 0 Occipital bone, reduced ossification Fetuses affected 0 Litters affected 0

2

70 (17)

4

8

16

70 (17)

34 (18)

0 (0)

0 0

0 0

4 4

_ _

0 0

2 2

6b 6

_ _

111 (17)

119 (17)

54 (18)

7 (6)

13 9

0 0

19 9

11 6

2 2

0 0

5” 4

0 0

4 1

f 1

2 1

0 0

0 0

2TC 11”

36” 18”

T 6’

0 0

0 8

6” 5

6’ 6’

0 0

0 0

6” 5

6’ 6’

a-h’ Significantly different from controls (P < 0.05; P < 0.01; P c 0.001, respectively).

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DISCUSSION

The present study shows that S.C. MnCl, administration to mice during organogenesis causes maternal and developmental toxicity. The major indications of maternal toxicity were deaths (at 16 mg/kg/day), decreased weight gain and food consumption (at 8 and 16 mglkglday), and increased kidney weight and decreased liver weight at 16 mg/kg/day. Significant reduction in maternal body weight gain during pregnancy (P < 0.05) compared to control, treatment-related toxicologic signs in behavior, abortion, and death have been reported to be general clinical criteria for the existence of maternal toxicity [25,26]. At maternally toxic doses (8 and 16 mg/kg/day) and below (4 mg/kg/day), there was evidence of embryofetotoxicity, expressed as a dose-dependent increase in resorptions per litter, a dose-dependent decrease in the number of live fetuses per litter, reduced mean fetal body weight per litter, and a dose-dependent increase in the incidence of skeletal variations. However, embryofetotoxicity was especially remarkable in the 8 and 16 mg/kg/day groups. It has been reported that manifested maternal toxicity also invariably causes reduction in fetal body weight, increased resorptions, and rarely fetal deaths [25,27]. Although some embryofetotoxic effects were also observed at 4 mg/kg/day, embryolethality was probably a direct consequence of the maternal toxicity. On reviewing 234 studies of agents tested in hamsters, mice, rats and rabbits, a fairly strong association between maternal toxicity and embryo/fetal mortality could be observed [26]. The results of the present study agree with previous investigations in which laboratory animals were given large doses of Mn during pregnancy [20&22,28,29]. In these investigations, there was no evidence of structural malformations in the offspring. Even direct i.p. exposure during the organogenic period in hamsters, although establishing an embryolethal effect, did not have a teratogenic effect [19]. Unlike the fetus, it has been reported that neonate seems to be particularly vulnerable to high levels of Mn [29]. With regard to workers exposed to Mn, it was reported that the number of births occurring during the exposure period was significantly lower than that expected on the basis of the fertility experience of a matched unexposed group [14]. However, in a recent study no effect of Mn exposure on fertility of male workers from a dry alkaline battery plant was evidenced [30]. It has been suggested that the workers participating in the first investigation [ 141 absorbed more Mn than those in the recent study [30], because they were exposed to various Mn salts which are more soluble in water than MnO,. In the present study, the no observable adverse effect level (NOAEL) for maternal toxicity in mice was 4 mg MnCl,.4H,O/kg/day. The NOAEL for embryotoxicity was 2 mg/kg/day, while there was no evidence of major malformations at any dosage level employed in this investigation. It has been reported that a Mn intake of 35 ,uglkglday should be adequate to maintain Mn balance for most individuals [31]. According to this value, and based on oral Mn intake, it is unlikely that the presence of Mn in the

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