Drinking water sources in the Rift Valley of Ethiopia contain fluoride (F) levels exceeding the World Health Organization (WHO) limit of 1.5 mg/L [1,2]. As a consequence, many people living in the region are severely affected by fluorosis [2,3,4,5,6,7,8]. In serious cases, dental fluorosis is manifested as brown mottling of the enamel and results in overall yellowing of the teeth with erosion of the enamel [2,8]. Skeletal fluorosis occurs when there is a high degree of bone brittleness due to excess deposition of F in bone that leads to osteosclerosis [3,6,9,10,11,12]. Non-skeletal fluorosis includes muscle weakness manifestations, such as stiffness of the back and neck muscles and pain leading to inability to carry out routine domestic activities [2,9].

Fluorosis is a major public health concern particularly for developing countries including Ethiopia, as the infrastructure needed to remove the excess F ions is lacking or not widely accepted [13,14]. It can affect both children and adults [13]. Over 85% of Ethiopians living in the Rift Valley have been exposed to excess levels of F intake [7,15,16]. Although drinking water is the dominant pathway of F exposure in Ethiopian Rift Valley areas, food stuffs prepared with high-F cooking water is also an additional pathway [17]. Foods added an average of 2.3 to 4.8 mg/kg F depending on village location and type of foods consumed, both plant and animal foods are locally grown in this high-F environment [2,17].

Epidemiological studies of communities with similar F exposures have shown a relationship between calcium (Ca) intake and reduction in severity of dental fluorosis [2,17,18,19]. The Ca binds with F forming an insoluble Ca fluoride complex in the gastrointestinal tract, preventing absorption and reducing the extent of F exposure [20]. In this way the adverse effects of F are decreased. Two animal studies have shown that proof of principle [20,21]. To our knowledge, only one study has reported an inverse association between dietary Ca (milk) and severity of dental fluorosis in individuals in Ethiopia [4] and no one has reported the association between Ca intake and the severity of skeletal and non-skeletal fluorosis.

In many areas of Ethiopia, dietary Ca intake, particularly of child-bearing women, is well below the recommended dietary allowance [22,23]. F exposure may be an added concern for women’s bone and dental health where there is low Ca intake. Therefore, the purpose of this study was to assess the dental, skeletal and non-skeletal fluorosis symptoms and associations with dietary Ca intake among women of child-bearing age in the Ethiopian Rift Valley.

More than half of the women in this study had dental fluorosis as diagnosed using Dean’s index criteria [28]. This is in line with the findings of a recent systematic review which reported a high prevalence of dental fluorosis in the Ethiopian Rift Valley [7]. In addition, a high prevalence of dental fluorosis was reported among children living in moderate F (24.1%) and high-fluoride (75.9%) areas of the Ethiopian Rift Valley [13]. Another study also reported a higher prevalence of dental fluorosis (62%) among adult inhabitants in the main Rift Valley of Ethiopia [4]. Our data show that women of child-bearing age can be affected by high F, but dental fluorosis develops mainly during early childhood [31]. In contrast with Kravchenko et al. [4], who reported an inverse association with milk intake in young adults living in the Rift Valley, we found no predictors of dental fluorosis. This could be due to the fact that dental fluorosis is an irreversible process that occurred as a result of long-term exposure to excess fluoride during childhood period, and thus was no longer related to current calcium consumption.

Few studies have reported on non-dental fluorosis symptoms in women. We classified symptoms according to skeletal rigidity and pain (skeletal fluorosis), muscular weakness (a major outcome of non-skeletal fluorosis) and gastro-intestinal complaints (other symptoms of non-skeletal fluorosis [9]. Skeletal rigidity accompanied by joint pains was common among women. Muscular weakness manifestations were also observed in many of the women. In addition, early signs of F toxicity, such as gastro-intestinal complaints manifested as loss of appetite, nausea and constipation, were observed in some of the women.

The women in the present study had a low intake of dietary Ca, averaging close to 400 mg per day. This is in agreement with the findings of Tesfaye et al. [22] who assessed national Ca intakes of Ethiopian women in the reproductive age using a single 24 h dietary recall and found the national average to be 478 mg per day. For women in the same state as our study, the Southern Nations, Nationalities and People Regional state (SNNPR), these authors found average Ca intake of 622 mg per day. In contrast, the Ca intakes of women in this study were estimated from daily and weekly consumption frequencies of Ca-rich foods during a one-month period. Differences may be due to different methodologies or actual differences in our kebeles; however, both sets of results show a lack of adequate dietary Ca in most women.

In our regression analysis, study mothers who had inadequate Ca intake (?400 mg/d) had three times increased odds of developing skeletal and non-skeletal fluorosis symptoms, namely skeletal rigidity with joint pains and muscular weakness manifestations, compared with those with higher Ca intake. To our knowledge, no study has been conducted to assess Ca intakes as predictors of skeletal or non-skeletal fluorosis symptoms. Kravchenko and colleagues [4] found a significant correlation between milk intake and dental fluorosis severity among men and women in the Ethiopian Rift Valley. Therefore, milk was the main source of Ca and presumably measured intake represented usual practice. Not many women in our study ingested dairy products as a source of Ca.

A decrease in F absorption by dietary Ca has only been demonstrated in a small number of animal studies [20,21]. In these experiments, Ca added to the diet reduced urinary F excretion, indicating the gastrointestinal binding of F ions by Ca. Kebede et al. [20] also verified this finding by showing a decrease in fecal F levels. However, most support for the Ca effect on F has been shown in observational studies. An earlier epidemiological study of children in India reported greater prevalence of fluorosis symptoms among children with inadequate dietary Ca intake [18]. In that study, children with adequate dietary Ca (>800 mg/d) and deficient dietary Ca (<300 mg/d) having comparable intakes of F (mean 9.5 ± 1.9 mg/d) were compared; severe toxic effects of F were observed in children with Ca deficiency [18].

Similarly, our recent efficacy trial provided a proof of concept on calcium intake and fluoride. In this trial, the study subjects were supplemented with an approximate of 1000 mg of calcium (using calcium-containing eggshell powder) on a daily base for six consecutive months. The urinary F excretion in the supplemented group was six-fold lower (? = ?6.1 (95% CI: ?7.1, ?5.1)) compared to the control group. The risk of developing skeletal and non-skeletal fluorosis was also significantly (p < 0.001) reduced in the treatment group [23].

Gastrointestinal complaints (Index-3) were not associated with Ca intake, or any other factors related to development of fluorosis, in contrast to skeletal rigidity and pains (Index-1) and muscular weakness (Index-2). Assefa et al. [3] used four clinical signs of non-dental fluorosis and compared these to radiological confirmation of skeletal fluorosis in 180 men and 5 women in Ethiopia from a high F area, mean age 55 years. Skeletal fluorosis was present in 70% of subjects, while clinical prevalence using kyphosis, impaired squatting, impaired neck mobility and impaired lumbar mobility, averaged 20%. This indicates clinical signs have low sensitivity; however, all signs except kyphosis were in high agreement (p < 0.001) with radiological skeletal fluorosis, indicating good specificity.

In the current study, older age was significantly associated with skeletal and non-skeletal fluorosis symptoms. This is in line with the findings of a community-based survey on epidemiology of skeletal fluorosis by Melaku and his colleagues [5]. These authors reported that older people of age 55 years and above had 20 times higher risk of developing skeletal fluorosis than adolescents and young adults of 15 to 24 years of age [5]. This could be due to the fact that in an area where F intake is higher, more F would be accumulated in bones and soft tissues of individuals who consumed it throughout their longer lifetime.

Our findings showed that women that have a high parity had greater odds of developing skeletal and non-skeletal fluorosis symptoms compared with those with lower parity. Older women were found to have a greater number of children, and hence parity may reflect the older age of women with more children and additionally, the stress of repeated pregnancy and lactation that may have depleted the Ca stores of these women [32,33]. Consequently, fluorosis symptoms would be manifested more among the women with high parity level. Therefore, this finding may have implications for recommendations of Ca during pregnancy and lactation in high-F areas.

The F level in drinking water sources in this area averaged ~5 mg/L and in staple foods ranged from 0.8–13.6 mg/kg. Similarly, the F level of the drinking water in this area was previously reported as 4.6 ± 1.7 mg/L [2]. The primary preferred option may be to find a supply of safe drinking water with safe F levels. In such instances, de-fluoridation may be sought as a solution [34,35]. However, there was no defluoridation of high-F containing water as well as no fluorosis prevention strategies attempted in the current study area. In this area, rainwater, a source of low-F drinking and cooking water, was not used for drinking as it was scarce and due to fearing of dust contamination from the roof of the house when available. Rainwater is not viewed as clean in many parts of the world [36]. In terms of addressing fluorosis symptoms, new technologies may assist, for example, in remineralization of teeth [37].

The limitations of this study include using some non-specific clinical symptoms and physical exercise tests to assess skeletal and non-skeletal fluorosis may be inaccurate and may introduce mis-classification bias. However, we employed the same dentist and physiotherapist to do all measurements, which reduces measurement bias. We also did not include women with only one symptom as being fluorotic to minimize the false positives arising from counting one nonspecific symptom. For skeletal fluorosis, we did not have access to x-ray measurements. We combined symptoms to create unique indices in order to analyze the presence of multiple symptoms. Daily dietary calcium intake of the women was estimated from frequencies of calcium-rich foods consumption which might not have estimated the usual calcium intake of women beyond that month.

Signs and symptoms of dental, skeletal and non-skeletal fluorosis were prevalent in women of child-bearing age in this area of the Rift Valley of Ethiopia, where water and food sources of fluoride were high. Dietary calcium intake was low relative to requirements, averaging only 400 mg per day. The low intake of dietary calcium, less than 400 mg per day, was significantly associated with musculo-skeletal fluorosis symptoms but not with non-skeletal fluorosis or dental fluorosis. The presence of musculo-skeletal fluorosis impairs everyday life for the women, who have a very labor-intensive life of raising children and having farm and home responsibilities. This suggests the need for further investigation on improving calcium intakes to mitigate the toxic effects of high fluoride intake in the Ethiopian Rift Valley.