Lead exposure inhibits fracture healing and is associated with increased chondrogenesis, delay in cartilage mineralization, and a decrease in osteoprogenitor frequency

Environmental Health Perspectives, June, 2005 by Jonathan J. Carmouche, J. Edward Puzas, Xinping Zhang, Prarop Tiyapatanaputi, Deborah A. Cory-Slechta, Robert Gelein, Michael Zuscik, Randy N. Rosier, Brendan F. Boyce, Regis J. O'Keefe, Edward M. Schwarz

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice m various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification. Key words: endochondral ossification, fracture healing, lead, osteoblast, osteoclast, Pb, Pb toxicity. Environ Health Perspect 113:749-755 (2005). doi:10.1289/ehp.7596 available via http://dx.doi.org/[Online 14 March 2005]

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Despite stringent governmental regulations, lead exposure continues to be a major public health problem, with blood levels being elevated in a bimodal distribution predominantly affecting young children 1-5 years of age and individuals > 50 years of age (Pirkle et al. 1998). The Centers for Disease Control and Prevention's (CDC's) Childhood Lead Surveillance Program monitors blood Pb at the state and local levels. Mean national blood Pb levels have decreased dramatically over the past 30 years, as documented by the third National Health and Nutrition Examination Survey, Phase 2 (NHANES, 1991-1994) (CDC 2000; Pirkle et al. 1998). According to NHANES data from 1999 and CBLS data from 1996-1998, despite the decreases in mean national blood Pb among children, levels remain elevated in specific areas, affecting mostly low-income, urban children and those living in older housing (CDC 2000).

In addition to children, many adults maintain elevated blood levels due to past occupational or environmental exposure. With the reduction of Pb in fuel and soldered cans as well as increased awareness and vigilance, acute environmental Pb exposure has decreased dramatically (Pirkle et al. 1998). However, Pb becomes sequestered in the skeleton, incorporated into hydroxyapatite crystals during calcification, and remains there until the bone is resorbed or remodeled (Wittmers et al. 1988).

Elevated blood Pb levels, particularly perimenopausal, may have a causative role in the pathogenesis of the costly metabolic bone disease osteoporosis. Blood Pb levels increase during periods of high bone turnover such as menopause (Silbergeld et al. 1988, 1993). Additionally, the aging process itself has been shown to increase the release of Pb from the skeleton by cadaveric analysis as well as by experimental study (Barnes et al. 1999). In vivo models have demonstrated a decrease in bone density with Pb exposure (Escribano et al. 1997; Gruber et al. 1997). In addition, multiple reports in humans and animals support a role for Pb in osteopenia (Berlin et al. 1995; Silbergeld et al. 1988). The decrease in bone quality may not only cause individuals to cross a fracture threshold earlier, but, we hypothesize, it may also impede normal fracture healing.

It is widely appreciated that the role of the skeleton in Pb toxicokinetics is greater than that of a reservoir. Several authors have described the inverse relationship between elevated blood Pb levels and skeletal development, chest circumference, and stature (Pearl and Boxt 1980; Schwartz et al. 1986; Shukla et al. 1989). The effects on adults are more subtle. Previously, we identified chondrocytes as important targets of Pb toxicity (Puzas et al. 1992) and demonstrated that Pb suppresses the expression of phenotypic markers in growth plate chondrocytes (Hicks et al. 1996). More recently, we have shown that Pb alters the effects of parathyroid-hormone-related peptide, transforming growth factor-[beta] (TGF-[beta]), activator protein-1, and nuclear factor-[kappa]B signaling in chondrocytes (Zuscik et al. 2002). Histomorphometric studies have demonstrated a significant Pb-associated decrease in length of rat femoral growth plate cartilage (Gonzales-Riola et al. 1997). Together, these data suggest an inhibitory effect on endochondral ossification (Gonzales-Riola et al. 1997; Hicks et al. 1996).

Several authors have demonstrated adverse effects of Pb on both bone formation and resorption mediated by cellular pathways affecting osteoblasts and osteoclasts (Dowd et al. 1990; Long et al. 1990; Miyahara et al. 1994; Pounds et al. 1991; Schanne et al. 1989, 1990). Osteoblasts are known targets of Pb toxicity from in vitro studies with ROS 17/2.8 cells, which demonstrated suppression of alkaline phosphatase, type I collagen, and osteocalcin (Long et al. 1990). In addition, circulating levels of osteocalcin, which serve as markers of osteoblast activity and regulators of bone formation and remodeling (Ducy et al. 1996), are decreased in Pb-intoxicated children (Markowitz et al. 1988). The mechanism by which these effects occur remains unclear.