Many organochlorine chemicals such as pesticides, herbicides and their intermediate products are widely distributed in the global ecosystem (Miyata 1993). People are regularly exposed to these substances, especially through consumption of meat and milk (Paul 1993). After absorption, these chemicals tend to accumulate in fat tissue and are retained for a long time without being metabolized (Philips et al. 1989, Yakusiji et al. 1984). Substantial amounts of such chemicals are found in human milk (Morita et al. 1975, Rogan et al. 1986, Wolff 1983).

Although placental and milk-mediated transfer of these stable lipophilic chemicals to fetuses and suckling offspring is an important route of exposure in humans, detailed data on tissue distribution and mother-fetus/suckling transfer of these chemicals are still sparse. The present study was performed to investigate the distribution of stable lipophilic environmental pollutants in rat dams exposed to a very small amount of hexachlorobenzene (HCB)⁴ and its transfer to their fetuses and suckling pups. In this experiment, we used HCB as an example of a stable lipophilic environmental pollutant. We examined its transfer from maternal organs to the F₁ generation animals at various stages of the prenatal and early postnatal periods. In Japan, pentachloronitrobenzene (PCNB) and pentachlorophenol (PCP), but not HCB, have been used in agriculture as pesticides. HCB is a by-product obtained during the production of these and other widely used pesticides and has been found as a contaminant in chlorinated pesticides (Kucher et al. 1969). It is a stable chlorinated hydrocarbon and has been detected in the placenta, maternal blood, milk and cord blood of humans (Ando et al. 1985). Chronic administration of HCB to animals results in a disturbance of porphyrin metabolism, hepatic degeneration and lipid peroxidation (Besten et al. 1993, Carlson and Tardiff 1976, Courtney et al. 1976). Besten et al. (1993) reported that female rats receiving a diet containing 0.03% HCB had significantly increased hepatic porphyrin accumulation and urinary porphyrin excretion from the 4th wk. However, rats treated with 0.015% HCB did not show these effects during the 4th wk but showed very slight increases after 10 wk. In the present experiment, pregnant rats were treated with the minimum level of HCB (35.1 nmol/100 g diet, 10 µg/100 g diet) that would subsequently produce a detectable organ concentration of HCB. Therefore, it was expected that the biological effect of HCB on the dams and newborns would be very weak. In this paper, we will describe the distribution of HCB in the organs of pregnant and nursing rats as well as its transfer to the fetuses through the placenta and to suckling pups through the milk.

MATERIALS AND METHODS

Table 1. Composition of the experimental diets [View Table]

On the day before parturition, the three pregnant rats in group 1 were anesthetized with ether and killed by cardiac puncture. Using heparinized syringes, blood samples were obtained, and the fetuses, placentas and organs were dissected and weighed. On d 2, 7 and 11 after birth, one pup each from the litters of dams in groups 2, 3 and 4 was similarly killed and its blood was collected. Organs and fat tissues were removed from each suckling rat and weighed. On d 16 after birth, the remaining sucklings and dams were similarly killed and the blood, organs and fat tissues were obtained. These collected samples were frozen immediately and stored at 20°C. All procedures were in accordance with the guidelines for the National Institute of Health and Nutrition.

Table 5. Concentration of hexachlorobenzene (HCB) in blood, organs and fat tissues of dams fed the diet containing HCB at 31.5 nmol/100 g diet before parturition (Experiment 2)1 [View Table]

Table 2. Body weights of pregnant and lactating rats fed the diet containing hexachrolobenzene at 35.1 nmol/100 g diet and those of fetuses and suckling pups in Experiment 11 [View Table]

RESULTS

Although the pregnant and nursing rats were fed the same HCB diet, the HCB concentration in the blood of pregnant rats on the day before parturition was about 2.5 times higher than that of nursing rats on d 16 after parturition (Table 3). On the other hand, the HCB concentration in the blood of suckling pups was 6.3 times higher than that of their dams. Because many organochlorine chemicals are most concentrated in fat tissue, we compared the HCB concentrations in the fat tissues between mothers and their sucklings. The HCB concentration in the subcutaneous and perirenal fat tissues of pregnant rats was about 14 and 1.8 times higher respectively, than that of lactating rats, and the concentration in the subcutaneous and perirenal fat of suckling pups was about 29 and 15 times higher, respectively, than that of their dams. Therefore, we concluded that a large portion of the HCB that had accumulated in dams during the pregnancy and nursing periods was transferred to their suckling pups.

Table 3. Concentration of hexachlorobenzene (HCB) in the blood and fat tissues of dams fed the diet containing HCB at 31.5 nmol/100 g diet and their fetuses and suckling pups in Experiment 11 [View Table]

The mean body weight of pregnant rats was naturally higher than that of three nursing groups (Table 4; P < 0.05). However, no significant differences in body weight were observed among the three lactating groups. Perirenal fat weight of the pregnant group was significantly higher than that of the three nursing groups (P < 0.05), but there were no significant differences among the nursing groups. No significant differences in liver, kidney or brain weight were observed among the four groups.

Table 4. Body and organ weights of dams fed the diet containing hexachlorobenzene (HCB) at 35.1 nmol/100 g diet before parturition and that of their fetuses and suckling pups in Experiment 21 [View Table]

In suckling pups on d 16 after birth, the body weight and brain weight of the group exposed to HCB both prenatally and postnatally were significantly lower than those of the groups exposed only prenatally or postnatally (P < 0.05) (Table 4). The liver weight of the group exposed to HCB both prenatally and postnatally was significantly lower than that of the group exposed only postnatally (P < 0.05). No significant difference in the perirenal fat weight was observed among the three suckling groups.

The HCB concentration was highest in the fat tissue and lowest in the brain of the pregnant rats on the day before parturition (Table 5). On d 16 after parturition, nursing groups fed the HCB diet during pregnancy had significantly lower concentrations of HCB in the blood, organs and fat tissue compared with the pregnant group (P < 0.01). Because HCB had rapidly disappeared from the body of the nursing rats during the 16 d of lactation, no significant differences in HCB concentrations in the blood, organs and fat tissue were observed among the three nursing groups.

Table 6. Concentration of hexachrolobenzene (HCB) in the placenta and whole fetus, liver and brain of fetuses of dams fed the diet containing HCB at 35.1 nmol/100 g diet in Experiment 21,2 [View Table]

HCB was detected in the nursing rats fed the HCB-free diet, although the level was low. To investigate the cause, we measured the concentration of HCB in the diets used in these experiments and in the pellet (the ingredients and pellet were commercially obtained from Japan Clea). The HCB concentration was 35.45 ± 0.27 nmol/100 g diet (n = 3) in the HCB diet, 0.020 ± 0.014 nmol/100 g diet (n = 3) in the HCB-free diet and 0.044 ± 0.007 nmol/100 g diet (n = 3) in the pellet. Therefore, we concluded that the small amounts of HCB detected in the body of the nursing rats fed the HCB-free diet was probably due to contamination of these diets by HCB.

Possible transfer of HCB from dams to fetuses was investigated using pregnant rats fed the HCB diet from d 2 of pregnancy to the day before parturition (group 1). The HCB concentrations in the carcass, liver and brain of the fetuses were lower than that in the blood of their dams (Table 5) and placentas (Table 6) (P < 0.05). Within the fetus, the HCB concentration was lowest in the brain (P < 0.05). Given an average body weight of the fetuses of 4.2 g, an average litter size of eleven (Table 4), and an HCB concentration in the fetus carcass of 10.13 pmol/g, the estimated amount of HCB transferred from a dam to her litter was about 468 pmol (43 pmol per fetus). Based on the food intake of pregnant rats from d 2 of pregnancy to the day before parturition of 343 ± 21 g, the intake of HCB from diet was 120 ± 7 nmol. Therefore, the amount of HCB transferred to the fetuses was estimated to be about 0.39% of the amount consumed by the dam.

To study the transfer of HCB accumulated during pregnancy from dams to their suckling pups through milk, the concentration of HCB in the stomach contents, blood, liver and subcutaneous fat of suckling pups on d 2, 7, 11 and 16 after birth were determined (Fig. 1). On d 2 after birth, the HCB concentration in the stomach contents was higher in the suckling pups fed by the dams exposed to HCB during pregnancy than in those fed by the dams who had not been exposed to HCB (P < 0.05). However, the concentration decreased gradually during the postnatal days and finally reached the same level as in the suckling pups fed by nonexposed dams. On d 2 after birth, a small amount of HCB was detected in the stomach contents of suckling pups that had postnatally received milk from nonexposed dams. In this experiment, the litters were exchanged 24 h after birth, between the dams having ingested the HCB diet and those having ingested the HCB-free diet during pregnancy. Therefore, the small amount of HCB found in the stomach contents of the suckling pups fed by the dams having ingested the HCB-free diet was due to the contaminated milk from the dams who fed them for the first hour. On d 2 after birth, the HCB concentration in blood of the suckling pups exposed to HCB both prenatally and postnatally was higher than that of suckling pups exposed either prenatally or postnatally (P < 0.05), but decreased gradually during the 16 d after birth. The HCB concentration of the suckling pups exposed to HCB only postnatally increased for 7 d after birth and then decreased gradually. However, in those exposed to HCB only prenatally, the concentration decreased gradually after birth. The hepatic HCB concentration was higher in the suckling pups exposed to HCB both prenatally and postnatally than in those of the other two groups on d 2 after birth. The hepatic concentration decreased gradually during the 16 d after birth. The HCB concentration of the suckling pups exposed only postnatally increased during the 7 d after birth and then decreased gradually. However, in those exposed to HCB only prenatally, the concentration decreased gradually after birth. In terms of subcutaneous fat tissue, the suckling pups exposed to HCB both prenatally and postnatally had the highest concentration of HCB, whereas those exposed to HCB only postnatally had an intermediate value, and those exposed to HCB only prenatally had the lowest value on d 7 after birth. In the three groups, the concentrations decreased gradually during the 16 d after birth.

From these results, we conclude that the HCB that accumulated in dams during pregnancy was transferred to their suckling pups during the early lactation period.

DISCUSSION

As described above, on d 2 after birth, a large amount of HCB was detected in the stomach contents of suckling pups fed by the dams that had ingested the HCB diet during pregnancy only (Fig. 1). On the contrary, HCB concentrations in the organs and fat tissue of nursing rats exposed to HCB during pregnancy were remarkably low on d 16 after parturition, and nursing rats fed the HCB (Table 3) and HCB-free (Table 5) diets showed lower HCB concentrations than pregnant rats. Results of these studies show that a greater portion of HCB in the dams was ingested by suckling pups through milk in the early days after birth. Because HCB concentrations were higher in suckling pups than their dams fed the HCB diet during pregnancy and lactation (Table 3), we concluded that a large amount of HCB in the whole body of nursing rats was transferred to and accumulated by suckling pups. In suckling pups, the highest concentration of HCB was found in the fat tissue. About 0.39% of HCB ingested by dams was estimated to be transferred to fetuses. Prenatal transfer to the fetuses was very small, but a large amount of HCB was transferred from dams to their sucklings through milk, resulting in rapid disappearance of HCB from dams during lactation. Abbott et al. (1968) and Quinby et al. (1965) reported that chlorinated compounds were detected in fat tissue of stillborn infants. This result suggests that fetuses are also exposed to lipophilic chlorinated compounds passing through the placental barrier.

HCB is stable and metabolized very slowly through a well-known pathway. A previous report has indicated the involvement of cytochrome P-450 3A in the microsomal oxidation of HCB to PCP and to tetrachlorohydroquinone (TCBQ) (Van Ommen et al. 1989). The report suggested that HCB, similarly to dexamethazone, induced cytochrome P-450 3A in rats (Besten et al. 1991). However, HCB is lipophilic and tends to accumulate and be retained in fat tissue for a long time without being metabolized. Rozman et al. (1977, 1981 and 1983) reported that the whole body half-life of HCB was ~3 mo in rats and 2.5-3 y in rhesus monkeys. In our study in which 10-wk-old nonpregnant female rats were fed a diet containing HCB at 31.5 nmol/100 g diet for 2 wk, the HCB concentrations in the epididymal fat tissue and blood were 2.64 ± 0.25 nmol/g and 5.65 ± 1.36 nmol/L, respectively (n = 6). After the HCB-free diet was given for the subsequent 4 wk, the corresponding concentrations were 1.48 ± 0.15 nmol/g and 5.26 ± 1.45 nmol/L, respectively (n = 6) (unpublished data). Thus, a 44% decrease in the HCB concentration in the epididymal fat tissue was observed after 4 wk of HCB withdrawal. In the blood, however, no significant decrease was observed after the 4 wk. Because HCB is excreted very slowly, we concluded that the HCB that had accumulated in dams during pregnancy was transferred to their suckling pups rather than metabolized by the dams or it was excreted in milk. To avoid harmful effects of HCB to dams and newborns, we used a dose which was very small but produced detectable concentrations in organs. We did not measure unchanged and metabolized HCB in the feces and urine. Further studies will be required to clarify the amount of HCB metabolized during pregnancy and lactation.

In Experiment 2, the HCB concentration in the stomach contents of suckling pups nursed by dams that had been fed the HCB diet was highest on d 2 after birth. On the other hand, the HCB concentration in their blood, liver and fat tissue increased during the initial 7 d after birth and then decreased to the same level as that of their mothers (Fig. 1). Thus, the decrease of HCB concentration in suckling pups was faster than the HCB half-life in adult rats reported by Rozman et al. (1977, 1981 and 1983) and that obtained in our study using nonpregnant female rats. These results are interpreted as follows: 1) HCB transferred from dams to their suckling pups through milk was rapidly metabolized; 2) HCB transferred to the suckling pups was rapidly excreted; and 3) HCB transferred to the suckling pups was diluted as a result of rapid growth. HCB has been shown to produce a toxic effect during prolonged exposure (Rozman et al. 1977). In the present study there was no evidence that the small amount of HCB harmed the dams. However, the suckling pups exposed to HCB both prenatally and postnatally had lower body weights than those exposed to HCB either prenatally or postnatally. Although breast feeding is carried out over a relatively short period of life, because infants are in a period of rapid growth and development, their susceptibility to toxic substances might be high. HCB has been reported to be hepatotoxic and immunotoxic, and to affect thyroid hormone homeostasis (Besten et al. 1993, Carlsom and Tardiff 1976, Rush et al. 1983, Vos et al. 1979). Although the present study did not confirm such negative effects, it is important to address the question whether prenatal and/or postnatal exposure to HCB produces long-term harmful effects. Further study will be required to determine the risk factors associated with the transfer and accumulation of these pollutants in breast-fed infants.

It has been reported that food restriction or starvation may decrease tissue concentrations of highly lipophilic compounds and accelerate their excretion (Dale et al. 1962, Wyss et al. 1982). We previously showed that there was little accumulation of newly absorbed pentachlorbenzene (PeCB) in fat tissue and organs and that it was rapidly excreted in rats given a restricted diet (Umegaki and Ichikawa 1990, Umegaki et al. 1993). We also observed that metabolism and excretion of lipophilic PeCB were markedly greater in rats fed a diet containing either fish oil or viscous dietary fiber than in rats fed control diets (Ikegami et al. 1991 and 1994, Umegaki et al. 1995). We concluded that this enhanced metabolism and excretion of PeCB were due to the small mass of fat tissue that resulted from these treatments. This findings support the idea that the large amount of HCB transferred from dams to suckling pups, which had very small amounts of fat tissue, was excreted immediately and rapidly after birth.

In conclusion, our study strongly suggests that although prenatal transfer of HCB to rat fetuses was very small, a great portion of the HCB that accumulated in dams during pregnancy was postnatally transferred to suckling pups through milk immediately after birth. Further study will be required to clarify the mechanisms which accelerate the metabolism and excretion of lipophilic environmental pollutants and depress their accumulation in the body during pregnancy and lactation.