Evaluation of calabash chalk effect on femur bone morphometry and mineralization in young wistar rats: A pilot study.

BACKGROUND: Calabash chalk, a popularly consumed geophagic material in Nigeria has been reported to contain lead, arsenic, alpha lindane, endrin, and endosulfan 11 among other pollutants. AIM: The continuous exposure of young children to this chalk necessitated this study on the bone morphometry and mineralization in young Wistar rats.
MATERIALS AND METHODS: Fourteen young (weanling) Wistar rats of both sexes weighing 54-72 g were assigned into two groups of seven animals each. Group I served as control, while group II was the test group (TG). 40 mg/ml of C. chalk was administered as suspension to the test animals in group II. Animals in the control group were orally treated with 1ml of distilled water. Administration of the C. chalk in the animals lasted for 28 days, and the animals were sacrificed on day 29, using chloroform anaesthesia. The femur bones were dissected out, cleaned of flesh and sun-dried. The lengths and weights of the femur bones were measured using graphite furnace atomic mass spectrophotometer.
RESULTS: Results showed 1.6% decrease in body weight change in the TG, insignificant decreases in the weights and lengths of both the right and left femur bones, and significant decreased (P < 0.0126) organ-somatic index, and femur bones concentrations (mg/l) of zinc, phosphate, carbonate, calcium, sodium, and potassium (P < 0.05).
CONCLUSION: In conclusion, this study showed that C. chalk may alter growth rate, and cause de-mineralization in the femur bone, hence, it may be detrimental to bone growth.

INTRODUCTION

The act of eating the earth including clay and chalk is neither new nor outdated.[1] Geophagia, the practice of eating earth is associated with religious beliefs, medicinal and dietary purposes. The implication of this act is the exposure to toxic substances and parasites that are present in the ingested earth.[2-4]

One of such geophagic materials is the Calabash chalk, consumed largely in West Africa as a remedy for morning sickness; hence it is highly patronized by pregnant women.[5-7] The chalk is found largely in Nigeria and other West African communities. It is also found in ethnic stores and markets in the United States of America and the United Kingdom. Different names have been ascribed to this chalk: Calabar stone in English, La Craie or Argile in French, Mabele by Lingala in Congo, Nzu by Igbo and Ndom by the Efik/Ibibio of Nigeria.[5-7]

The major component of C. chalk is aluminum silicate hydroxide from the kaolin clay group, with a possible formula Al2Si2O2(OH)4.[18] This chalk is available in varieties of forms including powder, molded shapes and blocks.[8]

Analysis of the C. chalk revealed some poisonous substances such as lead and arsenic.[68] Lead concentration in C. chalk has been reported to be approximately 40 mg/kg.[8] This concentration of lead in the C. chalk is far more than the approved dietary concentration.[9] Other toxic elements including aluminum, silicon, alpha lindane, endrin, and endosulfan 11 have also been reported.[8]

Research carried out with the chalk on rats revealed fragmentation of the parenchymal cells, and dilation of sinusoids of the liver due to treatment with the chalk.[10] Another report has it that it alters the normal concentration of hemoglobin, red blood cell counts and erythrocyte sedimentation rate (ESR), of female rats.[11]

Since there are reports that this chalk contains heavy metals and other toxic substances, the deposition of these substances in the bone tissue may result in bone disorders and poor growth.[12] Lead, a constituent of this chalk can be localized in areas of bone mineralization and growth.[13] Accumulation of lead in skeletal tissues begins during fetal development and continues throughout adulthood.[1415] It has been reported that lead adversely influences bone development through disruption of mineralization during growth.[16] As there are no clinical studies, it is important therefore to investigate the effect of C. chalk on the morphometry and mineralization of the bone in experimental animals with the aim of relating the findings to human situation, hence this study was conducted.

MATERIALS AND METHODS

Fourteen young (weanling) Wistar rats of both sexes weighing 54-72 g, obtained from the Animal House, Faculty of Basic Medical Sciences, University of Uyo, were housed in wooden cages with saw particles as bedding. The animals were handled in accordance with International regulation governing the use of laboratory animals. The rats were fed with normal commercial pellet (Vital Feed, Grand Cereal Ltd, Plateau State, Nigeria) and water was allowed ad libitum throughout the period of the experiment. The room temperature of the experimental animal house ranged between 26-29°C, with 12:12 hour light and dark cycle. The animals were allowed to acclimatize for 7 days before the beginning of the experiment. The animals were equally assigned into two groups. Group I served as the control group (CG), while group II served as test group (TG).

Non-salted Calabash chalk was bought from a local market in Ikot Omin, Calabar, Nigeria. The chalk was grounded to powder. 40 g of the chalk powder was dissolved in 1 liter of water to give a suspension which was stirred prior to administration. Animals in CG were treated with 1ml of distilled water, while those in TG were treated with 1ml of C. chalk suspension containing 40 mg/ml of the non-salted C. chalk. All administration was by oral intubation.

Treatment was given in the morning hours of 8-9 am and lasted for 28 days. On day 29, the weight of the animals was noted, and the animals were sacrificed by humane killing using chloroform anesthesia. The femur bones were dissected out, cleaned of flesh and sun-dried. The lengths and weights of the femur bones and their mineral contents were measured.

The weight of the femur bones was measured using a digital weighing balance to the nearest 0.01g. The length was measured from the head to the condyles of the femur, by means of a measuring tape to the nearest 0.1mm. The mean of each double reading was recorded for the weights and the lengths. The sum of the weight of both the right and left femur were taken as the organ weights, and the organ-somatic index of the femur bones in both groups was computed (Organ somatic index = Organ weight/body weight).

The femur bones were digested using analytical grade nitric acid (3 ml per tissue). Acid digest were diluted appropriately with double distilled-deionized water before analysis of the minerals. Bone phosphate (P) concentration was determined using the acid-molybdate method, and the absorbance read at 400 nm. The analysis for zinc (Zn) was done using the ZincoVer method and the absorbance was read at 620 nm. Sodium (Na), potassium (K) and calcium (Ca) were carried out using graphite furnace atomic mass spectrophotometer and absorbance read at 589 nm, 766.5 nm and 422.7 nm respectively. Mean values were obtained from two consecutive analysis.

Statistical analysis

Data were statistically analyzed using standard student t-test and the results presented as mean ± standard deviation. P value of <0.05 was regarded as significant.

RESULTS

On day 6 of administration, the fecal boluses of the test group (TG) animals were softer in consistency than those of the control group (CG) animals. On day 8, the color of the fecal bolus of the TG was found to be brown to milky color, and this fecal color was maintained till the end of the experiment, unlike the CG that had brownish brown color of fecal bolus. The feeding and water intake of the rats in both groups were not affected.

The mean initial body weights (g) of the TG animals were significantly lower (P < 0.0006) compared with the CG, while the mean final body weights of the TG animals were also significantly lower (P < 0.0008) compared with the CG. The mean change in body weight of the animals in the TG and CG were 52.17 g (47%) and 66.29 g (48.6%), respectively [Table 1]. The lengths of the femur bones in the TG were slightly shorter in both the right and the left sides compared to the CG. The differences were however not significant. There was also an insignificant decrease in the weight of both the right and left femur bones of the TG when compared to the CG [Table 2].

Table 1

Weight of animals of the control and test groups

Table 2

Weight and length of femur bones of the control and test groups

The organ-somatic index in the TG and CG was 0.0025 ± 0.008 and 0.0033 ± 0.004 respectively, with a statistically significant difference (P < 0.0126).

The mean femur bone concentration (mg/l) of Zn, P, Ca, Na, and K were significantly lower (P < 0.05) in the TG compared to the CG [Table 3].

Table 3

Minerals deposition in the femur bones of the control and test groups

DISCUSSION

Calabash chalk has been reported to contain lead, arsenic and persistent organic pollutants among other substances.[68] The mean concentration of lead as reported by Dean et al.[8] was approximately 40 mg/kg which far exceeded the European Union recommended safety level for dietary lead by 40 fold.[3] This and other heavy metals present in this chalk are likely to interfere with the normal process of bone formation.

In this study, we investigated the effect of the chalk on bone mineralization and other morphological changes in experimental Wistar rats. There was 1.6% decrease in body weight change of the test group (TG). This may result from disruption in nutrient absorption by the chalk, as kaolin, its major constituent, has been reported to coat the lining of the gastrointestinal tract.[17] As a result of this, the coating may have prevented the absorption of beneficial nutrients leading to poor nutrition, which ultimately resulted in a limited body weight gain reported in this study. Beside this, poor nutrition can also increase the risk to other adverse health effects.[18] It is reported that lead reduced body weight and bone mass.[19] As one of the poisonous substance found in the chalk, lead may have played a significant role in the body weight reduction observed in this investigation.

There were no significant change in the length and weight of each of the right and left femur bones of TG compared to CG, and no significant change between the right and left femur bones in all the parameter measures in both groups. This is consistent with the normal symmetry of the body. However, the organ-somatic index of the TG was significantly decreased. This may be due to the significant change in body weight observed in this study. Our study is at variance to a previous report.[19] Here lead treatment resulted in about 2% decrease in femur length.

Furthermore, the results of our investigation showed a significant decrease in the bone minerals content in TG, which may be due to the combined effects of lead and other toxic elements present in the C. chalk.[8] Also, the developing skeleton is much more sensitive to toxicity than that of the adult.[20] Lead adversely influences bone development through disruption of mineralization during growth.[16] Lead accumulates and substitutes calcium in bone tissues and the resultant effect is that of disruption of mineralization, alteration of compositional properties and bone formation mechanisms, as well as the gradual depletion of bone minerals.[21-23] Lead has also been implicated in reduction of bone strength,[24] oxidative stress, reduced Zn, Na, copper (Cu) and iron (Fe) in rat bone.[25] and impaired bone mineral density and content.[22] The reduction in bone mineralization could lead to an increase in bone fragility and osteoporosis. Our study is at variance to a report by Jamieson et al.[19] According to this report, femoral Ca and P concentration were not affected when treated with lead.

In conclusion, the effect of calabash chalk on femur bone morphometry and mineralization of young Wistar rats showed that C chalk may alter growth rate, and cause de-mineralization in the femur bone, and hence it may be detrimental to bone growth. As this is a case in rats, a clinical trial could be carried out on humans to determine its effects.