4
Biologic Markers of Lead Toxicity

In the last few years, considerable interest has developed in discovering and validating new biologic markers for many toxic substances. Identifying new biologic markers has helped scientists to understand much better the mechanisms of toxicity. This has also been the focus for biologic studies of the mechanisms of lead toxicity. Biologic markers are indicators of events in biologic systems or samples. The National Research Council (NRC, 1989a,b) has classified biologic markers into three types—markers of exposure, of effect, and of susceptibility. A biologic marker of exposure is an exogenous substance or its metabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell. A biologic marker of effect is a measurable biochemical, physiologic, or other alteration within an organism that, depending on magnitude, can be recognized as an established or potential health impairment or disease. A biologic marker of susceptibility is an indicator of an inherent or acquired limitation of an organism's ability to respond to the challenge of exposure to a specific xenobiotic substance. This chapter describes biologic markers of exposure, effect, and susceptibility for lead. It also establishes the biologic basis for the assessment of analytic techniques to monitor lead in sensitive populations, which will be described in Chapter 5.

BIOLOGIC MARKERS OF EXPOSURE

Any assessment of the toxicity associated with exposure to lead



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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations 4 Biologic Markers of Lead Toxicity In the last few years, considerable interest has developed in discovering and validating new biologic markers for many toxic substances. Identifying new biologic markers has helped scientists to understand much better the mechanisms of toxicity. This has also been the focus for biologic studies of the mechanisms of lead toxicity. Biologic markers are indicators of events in biologic systems or samples. The National Research Council (NRC, 1989a,b) has classified biologic markers into three types—markers of exposure, of effect, and of susceptibility. A biologic marker of exposure is an exogenous substance or its metabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell. A biologic marker of effect is a measurable biochemical, physiologic, or other alteration within an organism that, depending on magnitude, can be recognized as an established or potential health impairment or disease. A biologic marker of susceptibility is an indicator of an inherent or acquired limitation of an organism's ability to respond to the challenge of exposure to a specific xenobiotic substance. This chapter describes biologic markers of exposure, effect, and susceptibility for lead. It also establishes the biologic basis for the assessment of analytic techniques to monitor lead in sensitive populations, which will be described in Chapter 5. BIOLOGIC MARKERS OF EXPOSURE Any assessment of the toxicity associated with exposure to lead

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations begins with measurement of the exposure. In practice, one assesses lead exposure through environmental or biologic monitoring techniques to examine markers of exposure. Lead exposure is the amount of lead (from whatever source) that is presented to an organism; dose is the amount that is absorbed by the organism (NRC, 1990). Various factors—such as blood flow, capillary permeability, transport into an organ or tissue, and number of active binding and receptor sites—determine the path of lead through the body and can influence the biologically effective dose. For example, lead inhaled in dust could be retained in the lungs, removed from the lungs by protective mechanisms and ingested, stored in bone, or eliminated from the body via the kidneys. Toxicity can be observed in the kidneys, blood, nervous system, or other organs and tissues. At any step after exposure, biologic markers of exposure to lead can be detected. A key component of biologic monitoring of lead exposure is the toxicokinetic and physiologic framework that underlies such monitoring. Screening in the absence of knowledge about lead's in vivo behavior limits the interpretation of monitoring data for public-health risk. For example, if clinical management or regulatory actions are to be effective, the timing of lead exposure that is reflected in a typical blood lead value should be known, as should dose-response relations that link the body lead concentration with adverse health effects. Lead Absorption Humans absorb lead predominantly through the gastrointestinal and respiratory tracts. Little uptake occurs through skin, especially in nonoccupational exposures. Lead deposition and absorption rates in the human respiratory tract are complex functions of chemical and physical forms of the element and of anatomic, respiratory, and metabolic characteristics. Inhaled lead is deposited in the upper and lower reaches of the respiratory tract. Deposition in the upper portion leads to ciliary clearance of lead, swallowing, and absorption from the intestine. Smaller lead particles, especially those less than 1 µm in statistically averaged diameter, penetrate the lower, pulmonary portion of the respiratory tract and undergo absorption from it.

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations Human studies (Chamberlain, 1983; EPA, 1986a) have shown that about 30–50% of inhaled lead is retained by the lungs (the range reflects mainly particle size and individual breathing rate). These studies have used unlabeled lead aerosol (Kehoe, 1961a,b,c), radiolabeled oxide aerosol (Chamberlain et al., 1978), lead fumes inhaled by volunteers (Nozaki, 1966), ambient air lead around motorways and encountered by the general population (Chamberlain et al., 1978; Chamberlain, 1983), lead salt aerosols inhaled by volunteers (Morrow et al., 1980), and lead in forms encountered in lead operations, fumes, dusts, etc. (Mehani, 1966). Most (over 95%) of whatever lead is deposited in the human pulmonary compartment is absorbed (Rabinowitz et al., 1977; Chamberlain et al., 1978; Morrow et al., 1980). Thus, the overall rate of uptake is governed by lung retention (i.e., 30–50%). Uptake occurs rapidly, generally in a matter of hours. Evidence of complete and rapid uptake can be gleaned from analysis of autopsy lung tissue (Barry, 1975; Gross et al., 1975). The chemical form of inhaled lead appears to have little effect on uptake rate (Chamberlain et al., 1978; Morrow et al., 1980). Similarly, uptake is little affected by air lead concentration, even when it is greatly in excess of that commonly encountered in nonoccupational settings—up to 450 µg/day (Chamberlain et al., 1978). The above data apply to adults and are relevant for the sensitive adult population, i.e., pregnant women. In the case of children, no studies have experimentally documented rates of direct uptake of lead from lungs. On anatomic grounds (Hofmann et al., 1979; Hofmann, 1982; Phalen et al., 1985) and metabolic grounds (Barltrop, 1972; James, 1978), however, uptake in adults should be greater than uptake in children. In nonoccupationally exposed populations, lead uptake from the gastrointestinal tract is its main route of absorption. For adults, the lead content of foods, tap water, and other beverages is of main concern for lead exposure. For infants, toddlers, and older children, ingestion of lead-contaminated nonfood materials—e.g., dust, soil, and leaded-paint chips—is of additional concern. In some cases, such exposure can exceed that occurring through the diet (NRC, 1976, 1980; EPA, 1986a; WHO, 1987). Various studies of gastrointestinal absorption of lead in adults as derived from measures of metabolic balance (Kehoe, 1961a,b,c) and

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations isotope distribution (Hursh and Suomela, 1968; Harrison et al., 1969; Rabinowitz et al., 1974, 1980; Chamberlain et al., 1978) have documented that 10–15% of dietary lead is absorbed. The rate rises considerably, to as high as 63%, under fasting conditions (Chamberlain et al., 1978; Rabinowitz et al., 1980; Heard and Chamberlain, 1982). That suggests that lead in tap water and other beverages, which are often imbibed on an empty stomach, undergo higher uptake and pose proportionately greater exposure. Rate of lead uptake over the range of exposures likely to be encountered by the general population, up to at least 400 µg/day, seems similar (Flanagan et al., 1982; Heard et al., 1983). Studies of lead bioavailability in the human intestine (Chamberlain et al., 1978; Rabinowitz et al., 1980; Heard and Chamberlain, 1982) have indicated that common dietary forms of lead are absorbed to about the same extent. Lead sulfide in one study was absorbed to the same extent as other forms, and in another study was absorbed to the same extent with meals, but during fasting was absorbed less than the chloride. Particle size difference might account for the absorption difference between fasting and meals. Dietary lead absorption is considerably higher in children than in adults. Results of studies of both Ziegler et al. (1978) with infants and Alexander and colleagues (1973) with children indicate an absorption rate up to 50% from the intestinal tract. Young children ingest nonfood lead through normal mouthing behavior and particularly through the abnormal, excessive behavior called pica. Substantial uptake of lead and systemic exposure occur because of the high concentrations of lead in such media as dust, soil and leaded paint (Duggan and Inskip, 1985; EPA, 1986a; WHO, 1987); the ingestion of perhaps 100 mg, or even more, of such media (Binder et al., 1986; Clausing et al., 1987; Calabrese et al., 1989; Davis et al., 1990); and a bioavailability of up to 30% (Day et al., 1979; Duggan and Inskip, 1985; EPA, 1986a). The higher intestinal absorption of lead seen in developing versus adult humans is commonly observed in other mammalian systems, including nonhuman primates (Pounds et al., 1978) and rodents (e.g., Kostial et al., 1978). Percutaneous absorption of inorganic lead in nonoccupational populations is low. Moore et al. (1980) applied 203Pb-labeled lead acetate to intact skin of adult volunteers and obtained an average absorption rate

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations of only 0.06%. Lilley et al. (1988) applied lead as the metallic powder or nitrate salt solution to one subject's skin; it failed to increase the lead content of either whole blood or urine, but the lead content of sweat far from the area of application increased. Lead has long been known to cross the placental barrier in humans and other species and become lodged in fetal tissues (Barltrop, 1969; Chaube et al., 1972; Buchet et al., 1978; Alexander and Delves, 1981; Rabinowitz and Needleman, 1982; Borella et al., 1986; Mayer-Popken et al., 1986). The question of when lead begins to enter the fetus during maternal exposure is important, but has not been fully answered. Data of Barltrop (1969) and Mayer-Popken et al. (1986) suggest that lead entry occurs by the third or fourth month; data of Borella et al. (1986) and Chaube et al. (1972) suggest that uptake occurs later. Lead Distribution Absorbed lead enters plasma and undergoes rapid removal to various body compartments: erythrocytes, soft tissue, and mineralizing tissue. Removal occurs over a matter of minutes (Chamberlain et al., 1978; DeSilva, 1981). If exposure is constant, a steady state eventually occurs. Under steady-state conditions (i.e., stable exposure), plasma lead and erythrocyte lead are in equilibrium. The equilibrium fraction of lead in plasma is less than 1% and varies very little (Cavalleri et al., 1978; Everson and Patterson, 1980; DeSilva, 1981; Manton and Cook, 1984), but rises at blood lead concentrations of about 50 µg/dL or higher (DeSilva, 1981; Manton and Cook, 1984). Lead is removed from whole blood, under steady-state conditions, with a half-life that depends on such factors as total body lead burden, age, magnitude of external exposure, and the method of measuring half-life (according to total circulating lead or absorbed exogenous fraction as measured by isotopic tracer). Whole-blood lead measured with various protocols of experimental exposure has been found to have a half-life of about 25 days (Griffin et al., 1975; Rabinowitz et al., 1976; Chamberlain et al., 1978). That refers to the first, or short-term, component of blood lead decay. Actual measurements of half-life, commonly obtained through blood

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations lead changes that occur with reduction in chronic exposure, yield various and generally much higher values than those obtained experimentally; actual measurements reflect a larger contribution of a long-term component, described as many months in half-life. Early studies by Barry (1975, 1981) and Gross et al. (1975) showed that lead in most soft tissues is usually below 0.5 parts per million (ppm); age-dependent accumulation in kidneys (Indraprasit et al., 1974) and aorta (Barry, 1975; Gross et al., 1975) in nonoccupational populations has been reported. Available data are not sufficient to show whether soft tissue concentrations have been declining in response to lower air and dietary lead uptake in recent years. Such changes would be registered more readily in the youngest segments of the population, where cumulative body burdens are smaller. Studies that showed no lead accumulation with age in many soft tissues have been cross-sectional and theoretically would disguise the moderate accumulation that can occur with age but be offset by declining lead exposure in recent years. The human brain, the principal target organ of lead exposure, has low concentrations of lead—less than 0.2 ppm (wet weight)—on a whole-organ basis when there has been no occupational exposure. Lead content can rise by a factor of several in people with high lead exposure (Barry, 1975). In subjects with lethal poisoning, whole-brain concentrations are above 1 ppm (Okazaki et al., 1963; Klein et al., 1970). Region-specific distribution of lead in the brain has been documented. The highest concentrations are in the hippocampus and frontal cortex (Okazaki et al., 1963; Niklowitz and Mandybur, 1975; Grandjean, 1978). Barry (1975, 1981) showed that tissue lead concentrations were lower in infants than in older children. Those in older children were not materially different from those in adult women. A large body of laboratory and clinical evidence shows that lead accumulates with age in human mineralizing tissue, i.e., bones and teeth. Accumulation appears to begin at birth (or even in utero) and continues until the age of 50–60 years, when it starts to decrease through some combination of dietary, metabolic, and hormonal changes (CDC, 1985; EPA, 1986a; Drasch et al., 1987; Drasch and Ott, 1988;

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations Wittmers et al., 1988). Total lead content in bone can reach 200 mg in nonoccupationally exposed adults and much higher in those occupationally exposed to large concentrations. Drasch and Ott (1988) have confirmed that bone lead is cumulative at least from birth. Autopsy samples from infants less than 1 year old had a bone lead concentration half that of preschool children (0.33 vs. 0.62 ppm wet weight) and one-fifth that of people 10–20 years old (1.76 ppm). All bone types—cortical bone, such as midfemur, and trabecular bone, such as temporal bone and pelvis—were shown to accumulate lead, but the denser cortical bone had markedly higher concentrations in the two older groups. A sex-based difference in bone lead accumulation was observed in the oldest group for trabecular bone, males having statistically higher concentrations (p < 0.05). Recent measurements of bone lead in adult autopsy samples also documented continued accumulation in adulthood up to at least the age of 50 (Drasch et al., 1987; Wittmers et al., 1988). In the work of Drasch et al. (1987), temporal bone showed age-dependent accumulation throughout adulthood, including the 70s, whereas midfemoral and pelvic samples showed a plateau in middle age and then a decline. The latter decline was pronounced in females and was attributed to osteoporotic changes. Those data support the finding in analysis of NHANES II results that menopausal women have higher blood lead than younger women (Silbergeld et al., 1988). Men were estimated to have a significantly higher total skeletal lead burden than women—mean, 41.4 mg versus 24.1 mg. Comparison of recent analyses with data gathered 10 years earlier in the same laboratory and with identical methods indicated a marked decline of lead in femoral and pelvic samples across adult age groups, amounting to 30–50%. In similar investigations, Wittmers et al. (1988) examined tibia, skull, rib, ilium, and vertebra from 134 hospital autopsies for lead content as a function of age, lateral and cross-sectional analytic symmetry, and bone composition. Lead content was symmetric in positional location, but not bone type. Lead concentrations rose with age in all sample types, and there was some longitudinal variation within a bone specimen, but not enough to preclude use of single measurements in bone analysis.

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations Lead Retention and Excretion EPA (1986a) and ATSDR (1988) analyzed the retention and excretion of lead in humans and animals. Ingested lead that is not absorbed is lost through urinary and fecal excretion. Absorbed lead that is not sequestered in bone or some soft tissues is eventually eliminated through the kidneys or through biliary clearance into the intestine. Deposition in keratinizing tissues (nails and hair) is a minor elimination pathway. On the basis of various experimental measurements (Kehoe, 1961a,b,c; Rabinowitz et al., 1976; Chamberlain et al., 1978), the following can be said: Urinary loss of lead in adults makes up about two-thirds of total elimination. Fecal lead loss (of lead arising from biliary elimination—i.e., endogenous fecal lead) makes up about one-third. About 8% of the total is eliminated through Hair and nails. Whole-body lead elimination over the short term removes about 50–60% of the newly absorbed lead, with a half-life in adult volunteers of about 20 days (Rabinowitz et al., 1976; Chamberlain et al., 1978). Of the deposited fraction, 50% (i.e., 25% of lead initially absorbed) is eventually eliminated. Infants and children retain 50% of ingested lead (Alexander et al., 1973; Ziegler et al., 1978). Infants (and perhaps preschool children) have slower elimination than adults (Thompson, 1971; Alexander et al., 1973; Chamberlain et al., 1978; Ziegler et al., 1978; EPA, 1986a). Lead elimination through urine might depend on concentration, as estimated by Chamberlain (1983) on the basis of results of studies that reported blood and urine values in adults. Whole-body lead retention in humans subjected to constant exposure is accounted for largely by skeletal accumulation. Interactions of Lead with Nutrients The toxicokinetics of lead in humans are affected by the metabolic and nutritional status of the exposed subjects. Nutrition and nutritional deficiencies are of prime concern in very young children, in whom

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations increased lead exposure is concurrent with deficiencies in many interactive elements, especially calcium and iron (see Markers of Susceptibility). Interactive relations for lead have been reviewed elsewhere (Mahaffey and Michaelson, 1980; EPA, 1986a). Various child and infant nutritional-status surveys have documented iron deficiency in children under 2 years old, especially those in low socioeconomic classifications. They are also the children with the highest prevalences of high body lead (Yip et al., 1981; Mahaffey et al., 1982a; Mahaffey and Annest, 1986). Similarly, various reports have shown a strong negative correlation between calcium intake and blood lead in children (e.g., Sorrell et al., 1977; Ziegler et al., 1978; Johnson and Tenuta, 1979) and adults (Heard and Chamberlain, 1982). In the analyses of Ziegler et al. (1978), the inverse association of blood lead concentration and calcium intake in infants was seen to extend into the low part of the range of adequate intake. Other nutrients that interact inversely with lead exposure are zinc (Chisolm, 1981; Markowitz and Rosen, 1981) and phosphorus (Heard and Chamberlain, 1982). Mathematical Models Over the years, a number of attempts have been put forth to provide a quantitative, mathematical model of the relation of lead in exposure media to total and toxicologically active lead in the body, the in vivo compartmentalization of lead in the human body, the relation of lead in target tissues and organs to likely biologic markers of exposure and toxicity, and even the relation of direct dose biologic markers to markers of early effect. Modeling approaches to metals in general are described by Clarkson et al. (1988), and specific reviews of lead biokinetic modeling are provided by Mushak (1989) and EPA (1986a). Models of in vivo toxicokinetics of lead differ greatly, both in their use of empirical data and in the types of lead exposure to which they are applicable. One can broadly group toxicokinetic models of lead into linear and nonlinear forms. We are interested here primarily in models that are applicable to low-concentration lead exposure.

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations Linear Models Rabinowitz and co-workers (1976, 1977) used stable isotopic-lead distribution analyses in adult volunteers to develop a three-compartment model of lead disposition. The kinetically discernible compartments were blood (the most mobile, containing 2 mg of lead), soft tissue (of intermediate mobility, containing 0.6 mg of lead), and bone (the largest, most stable, with a half-life of a decade or more and sequestering most total body lead). The modeling efforts of Kneip and co-workers (Kneip et al., 1983; Harley and Kneip, 1984) expanded earlier approaches to a multiorgan model that can be used to estimate deposition in children of different ages. Figure 4-1 shows the major components of the Harley and Kneip (1984) approach that depicts six tissue compartments. Table 4-1 presents age-dependent estimates of lead half-lives for bone, kidney, and liver reported by Harley and Kneip (1984) for the age range of 1–20 years. Bone lead in children 1–6 years of age has a half-life that is only one-third that in older children and only about half that in 8-year-olds. In contrast (and as expected), soft-tissue lead half-lives are independent of age. Age dependence (over ages 1–15 years) of tissue burdens of lead was also estimated (see Table 4-2). Blood lead peaks at 2 years and then declines gradually. Bone lead estimates show that lead concentration in 1-year-old infants is about 60% of that in 7-year-olds—not greatly at odds with the laboratory ratio of 1:2 based on autopsy samples for about the same age interval. The models referred to are for essentially steady-state exposure with associated complete mixing of the linked lead pools, and they use first-order kinetics. They are chronic-exposure modeling approaches and are not to be considered valid for acute lead poisoning. Nonlinear Models At low to moderate lead exposure, linear models of lead in humans appear to be as good as any other form of mathematical depiction. However, any model intended to be broadly applicable to higher exposures must account for the known empirical curvilinearity of blood lead as a function of some external lead concentration (e.g., in water or air) and multiple subpools of lead (e.g., in blood and in bone).

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations FIGURE 4-1 Linear toxicokinetic model of Harley and Kneip (1984). Model has six components, including initial extracellular space-gut compartments.  Coefficients (λ) of compartment entry and exit are as indicated. Source: Kneip et al., 1983.  Reprinted with permission from NeuroToxicology; copyright 1983, Intox Press. The nonlinear model proposed by Marcus (1985) is an attempt to accommodate data that show that plasma lead manifests a concentration-dependent equilibrium with erythrocytes in humans and that blood lead concentration is nonlinear over a broad range of exposure. Workplace exposures represent the high end of the predictive range. The model provides a rather good fit for data from studies by DeSilva (1981) and Manton and Cook (1984) of subjects exposed over a broad range.

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations Lead-Binding Proteins The presence of proteins with an avidity for lead in kidney and brain was discussed in Chapter 2. In the erythrocytes of variably exposed lead workers, there is a lead-binding protein that appears to be inversely linked to clinical manifestations of occupational lead intoxication; i.e., the higher the erythrocyte concentration, the more resistant the worker appears to be to overt poisoning (Raghavan et al., 1980, 1981; Lolin and O'Gorman, 1986). The protein's function is reminiscent of kidney cytosol lead-binding protein (e.g., Oskarsson et al., 1982; Goering and Fowler, 1985). Lolin and O'Gorman (1988) measured the protein in lead workers with various degrees of lead exposure. The protein was quantitated as two peaks, which suggested a heterogenous protein. It was present in erythrocytes from all lead workers, but absent from controls. The threshold for induction of the protein was about 38 µg/dL or less; that indicates some utility for monitoring in occupational exposures. Equally important, concentrations of the protein are significantly lower in people with clinical toxicity. Furthermore, those workers found that the concentration of the erythrocyte lead-binding protein was related to intensity of exposure (past and present), not its duration. That is also typical of inducible proteins. Lolin and O'Gorman have postulated that the protein is protective in its function and would play a special role in subjects who are particularly susceptible to lead's effect on, e.g., ALA-D activity. They had found earlier (Lolin and O'Gorman, 1986) that there is a type of ALA-D activity inhibition not seen in environmental or nonoccupational exposures. Such protection targeted to preservation of ALA-D activity is consistent with the findings of Oskarsson et al. (1982) and Goering and Fowler (1985) that rat kidney ALA-D activity is notably resistant to lead and that this is due to the presence of a kidney cytosolic lead-binding protein. The analytic data on both worker erythrocyte and rat kidney cytosol protein structure suggest metallothionein, as noted by Goering and Fowler (1985) and Lolin and O'Gorman (1988), but further work is required to establish this fact.

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations Metabolites beta-Aminoisobutyric acid (beta-AIB) is a normal degradation product of thymidine, a constituent of DNA. Unlike typical amino acids, it is actively secreted as a catabolic metabolite via the tubule. It is normally excreted at low rates in humans not exposed to lead (6 nmol/µmol of creatinine). Farkas and co-workers (1987) examined the concentrations of this metabolite in urine of workers occupationally exposed to lead and in marmoset monkeys experimentally exposed via tap water. In workers with a mean blood lead concentration of 64 µg/dL and a mean EP of 117 µg/dL, there was a tripling of urinary output of beta-AIB. No threshold was determined for excretion beyond the normal range. In monkeys, there was a dose-dependent increase in urinary excretion. Given the fact of beta-AIB's handling by the kidney, the increase in the metabolite is a marker more of DNA damage through increased degradation of thymidine to beta-AIB. Identification of Toxicity Mechanisms Markers of effect not only are useful in the screening of high-risk populations, but also help to establish the various molecular and cellular mechanisms by which lead imparts multiorgan toxicity in those high-risk populations. Inhibition of 1,25-Dihydroxyvitamin D Formation As noted elsewhere, lead exposure is associated with reduced blood concentrations of the hormonal metabolite of vitamin D, 1,25-(OH)2-vitamin D. Such reductions with blood lead concentrations of 33–55 µg/dL, furthermore, rival those seen in several disease states (Rosen et al., 1980; Mahaffey et al., 1982b; Rosen and Chesney, 1983). Consequently, reductions in this hormone at lower lead exposures are signaling

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations early metabolic disturbance. Reduced concentrations of the hormone indicate that lead has two mechanisms of adverse effect that potentially can operate in high-risk populations. The first concerns the toxic consequences of disturbance in the hormone-calcium relationship, and the second concerns the many roles played by 1,25-(OH)2-vitamin D beyond regulatory control of calcium function. A major mechanisms of cellular lead toxicity appears to be interference in calcium homeostasis and function (Chapter 2). Such interference occurs either directly, via lead-calcium interactions in the cell, or through impaired function of calcium as a second messenger due to disturbed regulation by 1,25-(OH)2-vitamin D (Rasmussen, 1986a,b; Pounds and Rosen, 1988). It implies a risk of impaired handling of vesicular intestinal calcium and intracellular calcium in bone cells. Calcium-based effects are broadly distributed as to tissue and system sites, including the vascular system and developing neural and bone tissue. As summarized in Table 2-3, other physiologic functions potentially can be altered by reduced concentrations of 1,25-(OH)2-vitamin D. They include parathyroid phospholipid metabolism, cyclic GMP production in skin fibroblasts, rental phosphate reabsorption, and differentiation and proliferation of diverse cell types. In addition, the division, communication, and cytostructural organization of many cell types are affected. Impairment of Heme Synthesis Heme is a prosthetic group for many functional proteins involved in cell function and survival, and its formation is obligatory for cellular functions in many tissues, especially blood-forming tissue, muscle, kidney, liver, and brain (EPA, 1986a). Evidence of an effect of lead on heme formation would constitute a far-reaching mechanistic clue to lead toxicity. Heme formation is an intramitochondrial process. Its inhibition, whether by inhibition of the intramitochondrial enzyme ferrochelatase or by impairment of intramitochondrial delivery of the iron atom to protoporphyrin, can be considered a marker of generalized mitochondrial toxicity of lead in heme formation for a large number of cell and tissue types.

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations BIOLOGIC MARKERS OF SUSCEPTIBILITY One factor that can enhance susceptibility to lead toxicity is nutritional status. Various nutritional factors have been shown experimentally to influence absorption and tissue concentrations of lead (Mahaffey, 1985). Although experimental diets can be manipulated to create a wide range of nutritional problems, factors of clinical importance are far more restricted. Those considered of greatest importance to young children, as well as many adults, are total food intake, frequency of food intake, and dietary intake of some trace minerals, notably calcium and iron (Mahaffey, 1985). Fasting adults have been reported to absorb a substantially greater fraction of dietary lead than nonfasting adults (Rabinowitz et al., 1980; Heard and Chamberlain, 1982). Comparable data on the effects of fasting on lead absorption by children and young nonhuman primates are not available. To assess the role of dietary calcium in absorption and retention of environmental lead, it is essential to recognize that effects reflect both acute and long-term variation in dietary calcium intake. Numerous studies with experimental animals fed diets low in calcium have established that calcium deficiency increases both tissue retention and toxicity of lead (Mahaffey et al., 1981). Long-term calcium deficiency produces physiologic adaptive mechanisms (Norman, 1990), including increased concentrations of various binding proteins and stimulation of the endocrine and regulatory systems that regulate the concentration of ionized calcium. Parathyroid hormone and 1,25-(OH)2-vitamin D are critical to regulatory control of calcium. Physiologic controls that react to changes in dietary calcium also affect the biokinetics of lead. Generally, calcium deficiency increases lead toxicity (Mahaffey et al., 1973), but it is not clear that the increase in toxicity occurs predominantly because of physical competition between calcium and lead for absorption. The mechanisms that produce changes in lead absorption as a function of calcium status are not well documented. Humans can adapt to a wide range of iron requirements and intakes (Cook, 1990). There are two basic principles of adaptation to differences in iron intake: Regulation of iron absorption is achieved by the gastrointestinal mucosa, and fractional absorption depends directly on the metabolic need for iron. How the gastrointestinal tract achieves

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations adaptation to change in dietary iron intake and change in iron requirement remains an unanswered question in iron metabolism (Cook, 1990). Iron deficiency increases tissue deposition and toxicity of lead (Mahaffey-Six and Goyer, 1972). Ragan (1977) demonstrated sixfold increases in tissue lead in rats when body iron stores were reduced, but before iron deficiency developed. The influence of iron status on lead absorption has been investigated in adult humans, but with different results (Watson et al., 1980, 1986; Flanagan et al., 1982). It is not clear whether the difference reflects the severity of iron deficiency, differences in analytic approach, or some other undefined factors. A high prevalence of iron deficiency occurs in infants, children, and adolescents because of the need to expand the body's iron pool for growth. Women have higher iron requirements because of menstrual blood losses. The iron requirement of a normal pregnancy is approximately 500 mg, which is distributed to the fetus, placenta, and expanded maternal erythrocyte mass. It is critical to recognize that the groups of people who have the highest environmental lead exposures are also at greatest risk of iron deficiency (Mahaffey and Annest, 1986). The greatest impact of iron deficiency is in young children, who develop defects in attention span that lead to learning and problem-solving difficulties (Lozoff et al., 1985, 1987; Pollitt et al., 1986, 1989). Data from a longitudinal prospective study in Yugoslavia (Graziano et al., 1990) show the combined effects of lead exposure and iron deficiency among pregnant women, infants, and children. Adverse effects of both conditions on the neurobehavioral and hematopoietic systems were found. The importance of iron status for low-income families has been recognized for decades. In the early 1970s, a special program for low-income women, infants, and children was begun in the United States. Extensive evaluation of the impact of a program of nutritional supplementation for women, infants, and young children has been reported by Rush et al. (1988a,b). Children's blood lead concentrations tend to be associated with families' provision of intellectual and sociologic support (Milar et al., 1980; Hunt et al., 1982; Stark et al., 1982; Dietrich et al., 1985). In one study, the scores of mothers on three Home Observation for Measurement of the Environment scales were significantly associated with cumulative blood lead concentrations in infants: maternal involvement

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations with child, provision of appropriate play materials, and emotional and verbal responsivity of the mother (Dietrich et al., 1985). Scores on such scales are loosely associated with socioeconomic status, although the prevalence of these risk factors varies substantially within all social strata (Pearson and Dietrich, 1985). The association between poor maternal support for the child and blood lead remains after other factors are controlled for (Bornschein et al., 1985). One intrinsic, biologic factor of growing interest, but that has received little attention, is the distribution in sensitive populations of genetic susceptibilities that potentiate health risk. Few specific biologic markers of susceptibility for lead have been identified. The nature of lead effects and their genetic potentiation (or attenuation) must be understood both qualitatively and quantitatively. The relative distribution of genetically susceptible segments of the population is of main concern when these segments suffer substantial lead exposure. For example, lead exposure and the hepatoporphyric genetic disorder acute intermittent porphyria both produce accumulation of potentially neurotoxic ALA in plasma and urine (e.g., EPA, 1986a) and show some neuropsychiatric responses. A second genetic disorder, that associated with ALA-D deficiency, might well be a special problem for children at high risk for lead exposure in urban areas of the United States (Astrin et al., 1987). According to available data, any increased, genetically based susceptibility to lead exposure or its adverse effects is based mainly on lead's effects on heme biosynthesis and erythropoiesis. There is genetic polymorphism for the heme-pathway enzyme ALA-D in human populations. That has been recognized for some time (e.g., Granick et al., 1973), but the molecular genetic basis of the phenomenon has now been described (Battistuzzi et al., 1981). Potluri et al. (1987) identified the gene site at chromosome 9q34. Two common alleles, ALAD-1 and the deficiency ALAD-2, with frequencies of 0.9 and 0.1 in Europe-based populations, give rise to three phenotypes: 1-1, 1-2, and 2-2. Heterozygotic (1-2) people have ALA-D activity of approximately 50% of normal, and severely deficient homozygotic (2-2) people have activity of approximately 2% of normal (Doss et al., 1979). Doss et al. (1982) reported that workers with moderate workplace contact with lead but manifest lead poisoning in the form of high erythrocyte protoporphyrin concentrations were found to be heterozygotic for ALA-D deficiency, and Doss and Muller (1982) described an acute

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations lead-toxicity response in a person with ALA-D deficiency and moderate lead exposure. Ziemsen et al. (1986) reported that the fraction of lead workers with high blood lead increased as ALAD-2 phenotypy increased. Several recent studies have documented that children apparently heterozygotic or homozygotic for ALAD-2 can be susceptible to lead effects. Rogan et al. (1986) examined a group of children in a large lead-screening  program and found that, independently of blood lead, children with ALA-D deficiency also had significantly high EP; results of further testing suggested a problem with the amount of the enzyme, rather than a biochemically defective form. Astrin et al. (1987) have, however, found that ALAD-2 in a large sample of lead-screened children in New York City was correlated with the relative frequency of high blood lead (over  30 µg/dL). In acute intermittent porphyria, there is significant inhibition of the activity of the enzyme porphobilinogen deaminase, which mediates the conversion of porphobilinogen to uroporphyrinogen I. That leads to accumulation of high concentrations of ALA in urine and other body fluids (Goldberg and Moore, 1980; Moore et al., 1987). In both overt lead poisoning and attacks of acute intermittent porphyria, there are pronounced gastrointestinal, psychomotor, and cardiovascular responses, which have led to suggestions that lead works its adverse neurologic effects through direct action, as well as through the heme pathway (Silbergeld et al., 1982; EPA, 1986a). Conclusive evidence that lead significantly exerts neurotoxicity through the heme pathway, via excessive production of neurotoxic ALA (EPA, 1986a), has not been forthcoming. Studies directed to the hypothesis have entailed large exposures to lead, and it is not clear that low-dose lead exposure would effectively synergize neurologic manifestations of the attack stage of acute intermittent porphyria. Increased lead exposure affects the liver in various ways (EPA, 1986a). One site of toxicity involves impairment of hepatic biotransformations of endogenous metabolites (e.g., Saenger et al., 1984) and impairment of the detoxification and activation of drugs and other xenobiotic substances via effects on P-450 mixed-function oxidase (Alvares et al., 1976; Meredith et al., 1978) and perhaps the carcinogen-activating P-448 complex. Evidence of genetically differentiated hepatic biotransformation and biodegradation capacity in the P-450 and P-448 systems has been reviewed by Parke (1987). It is already known

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations that genetic diversity has an impact on biotransformation of various drugs (Parke, 1987). Animal systems show genetic polymorphism in P-450—P-448 systems, but the requisite human studies of the underlying molecular genetics remain to be done. SUMMARY The absorption, distribution, retention and excretion of lead in sensitive populations from various sources affect both the biologic monitoring of lead exposure and toxic outcomes. The literature dealing with the quantitative aspects of lead toxicokinetics is extensive and supports a number of conclusions. Inorganic lead is absorbed principally from the lungs and gastrointestinal tract of humans. About 30–50% of inhaled lead is absorbed from the lower respiratory tract in adults; the proportion is greater in children. Lead in water is absorbed variably: in adults, 10–15% is absorbed after consumption of blood; in children, approximately 50% is absorbed after consumption of food. Under fasting or semifasting conditions, rates of absorption rise considerably for adults and probably for children. Lead is absorbed into plasma; with steady-state exposure, redistribution of 99% or even more to the erythrocytes occurs. Newly absorbed plasma lead is distributed metabolically to blood, soft tissue, and various compartments of bone, where longer-term storage occurs. It is critical to recognize that skeletal lead stores are continuously remobilized as part of the physiologic remodeling of bone that accompanies the growth process. Skeletal accumulation of lead occurs through life until the sixth or seventh decade, when lead loss from bone occurs. Among adults, lead is released from bone in response to reductions in exposure or to metabolic stresses and alterations in the skeletal system. In people not occupationally exposed, up to approximately 200 mg of lead can accumulate. In lead workers, much more accumulates. Reversal of accumulation is associated with either dietary changes or, especially in postmenopausal women, bone-mineral homeostatic changes. The latter contribute to the blood lead burden and complicate lead toxicokinetic compartmental analysis.

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations Lead is excreted by the kidneys and gastrointestinal tract (biliary clearance occurs). Urine is the dominant route in adults. Spontaneous lead excretion is variable and is affected by biologic processes that complicate analysis, but plumburesis associated with chelation probing or chelation therapy is extensive and diagnostically useful in lead poisoning. Whole blood lead is the most popular and most useful indicator of lead exposure in acute and subacute poisoning. Blood lead measurement is also the most widely used measure of chronic lead exposure. Blood lead reflects relatively recent exposure in young children who were not excessively and not chronically exposed in their earliest years; but heavily exposed children and adults have a blood lead concentration at any time that integrates recent and older exposures. With respect to quantitative inputs to a given blood lead content, recent contributions account for the major fraction in both children and adults. Recent input is associated with a quick component, and accumulated lead input is reflected in a much slower component, with a longer half-life (e.g., Schütz et al., 1987a). Older exposures come into play via lead release to blood. One result of this phenomenon is a life-long legacy of earlier exposures. The policy and biomedical consequences are obviously important. For example, one must take account of the extent of such contributions to blood lead concentrations in planning the extent of control actions and concomitant population responses to lead regulatory actions. At low-dose lead exposures, which induce effects that are of increasing concern, blood lead concentrations around 10 µg/dL or less must be monitored in various populations. About that concentration are distributions of blood lead concentrations—an aggregate variance, which is a complex integral of exposure, interindividual variance, and the scatter associated with laboratory methods. Of those factors, the magnitude of exposure and the quality of method refinements would be the most responsive to attempts at minimization. Individual differences in lead toxicokinetics are intrinsic to populations and not readily amenable to control. Plasma lead, if it were amenable to clean collection and measurement, would have considerable interpretive value for assessing population lead exposures. Plasma, of course, transports lead to target tissues and major repositories. Contamination and loss problems are formidable

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations for ready analysis of plasma in the typical clinical or epidemiologic research laboratory. Not only is contamination a problem for plasma analyses, but small rates of erythrocyte hemolysis would increase error. A hemolysis rate of 1% would double the plasma lead content, assuming a 1% equilibrium lead fraction in plasma. Furthermore, we still need to understand the toxicokinetics of plasma lead distribution for various exposure scenarios; that requires that acceptable methods be developed. Bone lead measurement is the best way to assess body lead accumulation in such populations as high-risk urbanized young children, and it signals to the analyst and the policy-maker how rapidly lead is accumulating in bone. The role of this measure is enhanced considerably when techniques for integrating lifetime exposure are used in tandem with serial measurement of blood lead concentration in variably exposed young children or pregnant women. Lead is released from bone in response to reductions in exposure or to metabolic stresses and alterations in the skeletal system. Lead appears in several compartments in bone, and these vary in their ability to release lead to the bloodstream. Furthermore, the known distribution characteristics of lead in bone compartments indicate that one can probe both long-term and shorter-term lead storage rates in bone spectrometrically. Such probing can be used both with populations in systematic development (children) and with populations in senescence (post-menopausal women). Some biologic markers of effects of large exposures to lead have been characterized qualitatively and quantitatively and have been validated for toxicity in human populations. The usefulness of sensitive indicators, such as inhibition of particular enzymes, at very low lead concentrations has the added advantage that genetic susceptibility in lead-exposed subjects can be monitored. This would be the case for at least ALA-D activity distributions in sensitive populations exposed to lead. The sensitivity of some potentially useful biochemical markers has yet to be determined. Effect markers have been extremely helpful in identifying mechanisms of toxic action and in developing a better understanding of the biochemical interactions of lead in humans and other living systems. Finally, little attention has been given to the identification of biologic markers of susceptibility. Current limited information does show that

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Measuring Lead Exposure in Infants, Children, and Other Sensitive Populations some children and other sensitive people could be predisposed to increased lead intoxication because of genetic disorders and nutritional factors. This is a subject of increasing importance to toxicologists and those responsible for public health.