Pb2+ exposure alters the lens αA-crystallin protein profile in vivo and induces cataract formation in lens organ culture
Introduction
Lead is a common environmental contaminant whose most prevalent environmental exposure route, since the banning of leaded gasoline in the United States for example, is through exposure to lead-containing paint dust (Davis et al., 1993). The inadequate lead abatement in low-income housing stocks (Rabito et al., 2003) coupled with the increased intestinal absorption of lead in children (Heath et al., 2003) leads to a disproportional representation of low to moderate-level lead exposure in poor children. Chronic low-level Pb2+ exposure affects approximately 3% of children in the United States (Meyer et al., 2003) and results in IQ deficits and increased aggressive behavior. Lead exposure inhibits heme synthesis and induces abnormal neurological symptoms which may lead to convulsions and death (Sanborn et al., 2002).
Acute lead poisoning (BPb > 80 μg/dL) produces clinical ocular symptoms including amblyopia, blindness, optic neuritis and atrophy, muscle paralysis and decreased visual function (Fox, 2003). Low- to moderate-level lead exposure in children, and developmentally exposed rats and monkeys, also results in visual system deficits including alterations in rod and cone cell function (Fox et al., 1997). A correlation between low-level developmental lead exposure in the first trimester and rod-mediated retinal function has also been demonstrated (Rothenberg et al., 2002). Specifically, retinal changes characterized by an increase in scotopic electroretinogram a-wave and b-wave amplitudes, a measure of rod and possibly Muller cell function, occur if gestational lead exposure is greater than 10 μg/dL in the first trimester of pregnancy. This new type of rod dysfunction appears to be linked to the gestational and retinal development stage of the exposure.
Children environmentally exposed to high levels of Pb2+ retain higher tibia and circulating blood levels of Pb2+ 20 years after exposure than do unexposed age matched individuals (McNeill et al., 2000). This same study cohort also possessed an association of elevated tibial bone lead concentration and poorer performance on the Mini-Mental State Examination equivalent to that of aging 5 years (Stokes et al., 1998). The association of elevated tibial and patella bone lead levels in middle-aged and elderly men following 20 years after identification of lead contaminated drinking water was documented in a subset population of the Normative Aging Study (Potula et al., 1999). An examination of cognitive function of a larger subset of the Normative Aging Study demonstrated a steeper decline in cognition with age, for elderly men with elevated patella bone lead, equivalent to aging 5 years (Weisskopf et al., 2004). An examination of a subset of the Normative Aging Study identified a strong association between elevated circulating and bone lead levels and the decline of renal function for middle-aged and elderly men (Tsaih et al., 2004). This effect was more pronounced in diabetics and hypertensive subjects (Hu et al., 1996). Together these epidemiological studies support the association of previous lead exposure with later neurological and cognitive deficits in both children and adults and raise the question of whether chronic lead exposure impacts the development of diseases of aging.
Cataract, the clouding of the ocular lens, is an age-associated disease which is responsible for over forty percent of the cases of blindness in the world. A recent epidemiological study based on the Normative Aging Study has linked elevated tibial bone lead levels to an increased risk of cataract formation (Schaumberg et al., 2004). In the same study, lead exposure in conjunction with a secondary insult increases the risk of cataract formation as demonstrated in epidemiological data from smokers with increased bone Pb2+ levels. When the data was adjusted for pack-years of smoking the odds ratio remained at 3 in high versus low bone Pb2+ levels.
Analysis of normal and cataractous human lenses has demonstrated that opaque lenses have accumulations of heavy metals including Pb2+ independent of type of opacity while clear lenses have non-detectible levels of lead (Shukla et al., 1996, Cekic, 1998a, Cekic, 1998b). Organ culture of lenses using 203Pb as a radiotracer demonstrated the accumulation of Pb2+ at the basement membrane which may also diffuse into the lens fiber cells (Grubb et al., 1986). Short-term exposures to lead have not resulted in cataract formation. However, previous studies have demonstrated that clinically relevant levels of Pb2+ intoxication in orally exposed rats result in alterations of the redox status and cause protein oxidation in the lens (Neal et al., 1998, Neal et al., 1999). In conjunction, lipid peroxidation is also observed which may lead to lens cell membrane abnormalities.
The lens is a continuously growing non-vascularized tissue composed of concentric layers of ribbon-like lens fibers and a single anterior layer of cuboidal epithelial cells (Rafferty, 1985). Mature fiber cells, which lack nuclei and other organelles, comprise the body of the lens. The lens fiber cell protein complement is composed of ∼90% lens abundant structural proteins, the crystallins (Spector, 1985). Alterations in the three dimensional structure of the crystallins, or in the hydration layer and the surface charge density, which alter the protein structural network may have disastrous consequences for lens clarity through increased light scattering (Bettelheim, 1985). α-Crystallin has a high sequence similarity to small heat shock proteins such as HSP27 and prevents heat and/or denaturing agent-induced aggregation of other proteins in vitro (Horowitz, 1992, Derham and Harding, 1999). This chaperone activity may be crucial in preventing formation of large protein aggregates which are known to contribute to aging-related cataract development.
These data have led to the hypothesis that oral Pb exposure may lead to alterations in the post-translational modification and degradation of lens crystallins. These processes are believed to be programmed and critical to the long-term homeostasis and functional integrity of the lens (Jimenez-Asensio et al., 1999); when aberrant they can contribute to cataract formation. The current study examined alterations in the two dimensional gel electrophoresis patterns following short-term in vivo Pb2+ exposure and documents alterations in the degree of post-translational modification of a primary lens structural protein and chaperone, αA-crystallin. The current study also explored whether mature rat lenses maintained in organ culture with Pb2+, a system used to assess the lenticular toxicity of many pharmacological compounds, would develop opacities.
Section snippets
Materials
Lead acetate, urea, DTT, ultra-low temp agarose, CHAPS, trifluoroacetic acid, α-cyano-4-hydroxycinnamic acid and sequencing grade trypsin were purchased from Sigma. IPG strips and IEF markers were purchased from Amersham. Bis–Tris gels, MOPS running buffer, SilverQuest and Colloidal Blue staining kits were purchased from Invitrogen.
Animal exposure
Fisher 344 rats (75–100 g) were obtained from Charles River, fed and watered ad libitum, and maintained with a 12 h light:dark cycle in the University of
Results
As demonstrated in Table 1, rats given 2000 ppm of Pb2+ for 5 weeks with 1 week of withdrawal had elevated blood Pb2+ levels. These levels are in the low-lead exposure range and would not generally be treated by chelation therapy in adults but would possibly be treated if present in a child. Interestingly, the whole lens showed no evidence of Pb2+ accumulation.
A visual inspection of the two dimensional gel electrophoresis protein spot patterns of lenses from in vivo control and Pb2+-treated rats
Discussion
Recent epidemiological evidence links low-level lead exposure with increased risk of age-related diseases decades after initial exposure. The mechanisms whereby lead triggers the development of diseases as diverse as hypertension and cataract have not been clearly identified. Oral Pb2+ exposure in rats increases lipid peroxidation and oxidation of lens proteins while depleting cellular stores of GSH (Neal et al., 1998, Neal et al., 1999, Dwivedi, 1987). These oxidative modifications in the lens
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