Skeletal fluorosis in marsupials: A comparison of bone lesions in six species from an Australian industrial site

Highlights

• Skeletal fluorosis is described in six species of marsupial, five of which are new records.

• Skeletal fluorosis lesions in marsupials are consistent with the range of lesions in eutherians.

• Hyperostotic lesion distribution varies with biomechanical differences in gait and mastication.

• Interaction between biomechanics and fluoride toxicology enables prediction of pathology.

Abstract

In this study we explored the prevalence, type, location and severity of skeletal lesions in six species of Australian marsupial (Macropus giganteus, Notamacropus rufogriseus, Wallabia bicolor, Phascolarctos cinereus, Trichosurus vulpecula and Pseudocheirus peregrinus) from high and low-fluoride environments. Lesions occurred to varying extents in all species, and lesion distribution varied with biomechanical differences in gait and mastication. Bone fluoride levels increased with severity of periosteal hyperostosis. The mean bone fluoride concentration of individuals lacking hyperostosis (across all species, from both high and low-fluoride environments) was 1100 ± 260 μg F⁻/g dry bone, compared to 4300 ± 1200 μg F⁻/g and 6300 ± 1200 μg F⁻/g in those with mild and severe grade hyperostosis, respectively. Multivariable modelling showed that the probability of observing a lesion varied across species, anatomical location, age and bone fluoride concentration (in a non-linear manner). The pathological changes reported in the marsupials are consistent with the range of fluoride-related lesions described in other mammals, and biomechanical differences among the studied marsupial species offer some explanation for the degree of interspecific variability in prevalence, type, anatomical location, and severity of the lesions.

Introduction

Fluoride is ubiquitous in the environment, arising from both natural and anthropogenic sources. Natural release of fluorides occurs via the weathering and dissolution of minerals, emissions from volcanoes and marine aerosols (WHO, 2002). Fluorides are also emitted by a variety of industrial processes, including coal burning for electricity generation, mining and manufacturing of non-ferrous metals (aluminium in particular), and the production of ceramics, fertilizers and pesticides (NPI, 2014).

Due to its strong electronegativity, fluoride has a high affinity for bone and other calcified tissues such as teeth (Kierdorf et al., 2016a). Chronic intake of excess amounts of fluoride results in various forms of pathological changes in the skeleton, referred to as skeletal fluorosis or osteofluorosis (Shupe et al., 1992). A rise in the plasma fluoride concentration of herbivorous mammals in high-fluoride environments occurs following the shift from milk to plant diet after weaning (Kierdorf et al., 2016b). Any bone formed during exposure to a high concentration of fluoride in extracellular fluid is histologically impaired (Craig et al., 2016) however, lesions vary with the amount, timing and duration of fluoride intake (Bock et al., 2007, Boivin et al., 1989, Chavassieux et al., 1991, Czerwinski et al., 1988, Shupe, 1980, Singh and Jolly, 1970, Turner et al., 1993, Yoichiro, 1974). Exposure of developing (or growing) bones to excess levels of fluoride increases bone mass, however, the biomechanical properties of the newly formed bone are altered (Craig et al., 2016, Everett, 2011, Søgaard et al., 1994). Fluoride appears to increase osteoblast numbers due to a mitogenic effect on osteoblast precursors, while at the same time exerting a toxic effect at the cell level, causing a decrease in mineral apposition rate and an increase in mineralization lag time (Boivin et al., 1989, Chavassieux et al., 1991). While fluoride uncouples the processes of bone resorption and formation in favor of bone formation (Chavassieux et al., 1991, Everett, 2011), there has been some controversy about whether increased bone formation precedes (Bauer, 1945) or follows (Brearley and Storey, 1970) increased resorption.

The skeletal lesions associated with chronic exposure to excess levels of fluoride include mild to marked periosteal or endosteal new bone formation (hyperostosis, either localized or generalized), osteosclerosis, osteopenia and osteomalacia (Craig et al., 2016, Shupe et al., 1992, Simon et al., 2014) as well as peri-articular osteophytosis, joint ankylosis and calcification of tendon and ligament insertions (Roholm, 1937, Weatherell and Weidmann, 1959, WHO. World Health Organisation, 2004). The degree of these pathological changes varies in different bones of an individual, being largely determined by the relative rates of growth, by biomechanical factors and by the extent of bone remodelling (Malcolm and Storey, 1971). Metabolically more active portions of bone, such as those exposed to intense mechanical loading during locomotion, chewing or respiration, have a higher content of fluoride and show comparatively more severe pathological changes than other parts of the skeleton (Shupe et al., 1984). Overt lesions of osteoflurosis and the associated clinical signs typically do not manifest until bone fluoride concentration exceeds a level of 4000 μg F⁻/g dry weight (Schlatter, 1970, Shupe, 1980, Grace, 2008, Turner et al., 1993, WHO. World Health Organisation, 2002). Osteofluorosis-associated lameness has been reported in domestic rabbits, Oryctolagus cuniculus (Brearley and Storey, 1970), cattle, Bos taurus (Roholm, 1937, Suttie, 1957), sheep, Ovis aries (Yasar and Yur, 2008), horses, Equus caballus (Shupe and Olsen, 1983), and wild mammals (WHO, 2002), including eastern grey kangaroos, Macropus giganteus (Clarke et al., 2006).

Post-cranial skeletal lesions associated with chronic fluoride toxicosis have been described in humans (Choubisa et al., 2001, Christie, 1980, Krishnamachari and Krishnaswamy, 1973, WHO. World Health Organisation, 2004), cattle (Roholm, 1937, Shupe et al., 1963), sheep (Botha, 1993, Roholm, 1937, Simon et al., 2014, Weatherell and Weidmann, 1959), horses (Shupe and Olson, 1971), dogs, Canis lupus familiaris (Bauer, 1945), mink, Mustela vison (Shupe et al., 1987), rabbits (Bock et al., 2007, Brearley and Storey, 1970, Malcolm and Storey, 1971, Weatherell and Weidmann, 1959), rats, Rattus norvegicus (Dunipace et al., 1995), guinea pigs, Cavia porcellus (Roholm, 1937), rhesus monkeys, Macaca mulatta (Reddy and Srikantia, 1971), fruit bats, Pteropus spp. and Rousettus aegyptiacus (Duncan et al., 1996), white-tailed deer, Odocoileus virginianus (Suttie et al., 1985), antelope, Litocranius walleri and Tragelaphus eurycerus isaaci (Lloyd and Stidworthy, 2011), and eastern grey kangaroos (Hufschmid et al., 2015). Mandibular lesions have been described in horses (Shupe and Olson, 1971), cattle, sheep, and pigs, Sus scrofa domestica (Roholm, 1937), rabbits (Brearley and Storey, 1970), cats, Felis silvestris catus (Weatherell and Weidmann, 1959), roe deer, Capreolus capreolus (Vikøren and Stuve, 1996), and red deer, Cervus elaphus, (Vikøren and Stuve, 1996, Schultz et al., 1998).

Osteofluorotic lesions may differ among species for a variety of reasons besides levels of fluoride exposure and stage of skeletal growth. Variation in the biomechanics of gait will result in differing mechanical forces acting on bones, and consequent stimuli for bone remodelling, throughout the skeleton (Carlson et al., 2013). Studies on subchondral bone radiodensity in marsupials have demonstrated that compressive loads borne through the hind limbs are more substantial in bipeds than in quadrupeds (Carlson et al., 2013). The biomechanical forces acting on skull bones while feeding will also be altered by differences in diet, anatomy and mastication style (Crompton et al., 2010). The degree of horizontal and vertical movement of the mandibles during mastication varies greatly between species, largely as a result of anatomical adaptations to diet, for example, due to dentition adapted for a cutting versus a crushing action (Lanyon and Sanson, 1986a). The relative forces contributed by relevant muscle groups also differ for species that have a fused or flexible mandibular symphysis (Crompton et al., 2010).

This study compares the distribution and severity of osteofluorotic lesions in six herbivorous marsupial species: three bipedal, hopping macropodids and three arboreal quadrupeds. We examined specimens from high and low-fluoride areas in south-eastern Australia, and analysed whether the anatomical location and severity of these lesions were related to differences in fluoride exposure, using bone fluoride content as a biomarker, and biomechanics. Specifically, we tested the hypotheses that i) the higher compressive loads in the hind limbs of the bipedal species result in a higher proportion of osteofluorosis lesions in the hind limbs compared to the forelimbs, and ii) that quadrupeds exhibit an even distribution of lesions between forelimbs and hind limbs. As part of this investigation we also studied the microscopic characteristics of the osteoflurotic lesions in both the limb bones and mandibles. In addition, we further tested the hypothesis that species with a fused mandibular symphysis show a higher prevalence of mandibular osteofluorotic lesions than those with a flexible symphysis.