Development of California Public Health Goals (PHGs) for chemicals in drinking water.

As part of a program for evaluation of environmental contaminants in drinking water, risk assessments are being conducted to develop Public Health Goals (PHGs) for chemicals in drinking water, based solely on public health considerations. California's Safe Drinking Water Act of 1996 mandated the development of PHGs for over 80 chemicals by 31 December 1999. The law allowed these levels to be set higher or lower than federal maximum contaminant levels (MCLs), including a level of zero if data are insufficient to determine a specific level. The estimated safe levels and toxicological rationale for the first 26 of these chemicals are described here. The chemicals include alachlor, antimony, benzo[a]pyrene, chlordane, copper, cyanide, dalapon, 1,2-dichlorobenzene, 1,4-dichlorobenzene, 2,4-D, diethylhexylphthalate, dinoseb, endothall, ethylbenzene, fluoride, glyphosate, lead, nitrate, nitrite, oxamyl, pentachlorophenol, picloram, trichlorofluoromethane, trichlorotrifluoroethane, uranium and xylene(s). These risk assessments are to be considered by the State of California in revising and developing state MCLs for chemicals in drinking water (which must not exceed federal MCLs). The estimates are also notable for incorporation or consideration of newer guidelines and principles for risk assessment extrapolations.

Malathion and malaoxon environmental levels used for exposure assessment and risk characterization of aerial applications to residential areas of southern California, 1989-1990.

Between August 1989 and July 1990, California conducted a Mediterranean fruit fly eradication project in southern California which included repeated aerial applications of malathion bait to urban areas where approximately 1.6 million people resided. Concern about the safety of these applications prompted the California Department of Health Services to prepare a risk assessment. The current work presents the estimates of environmental levels of malathion and malaoxon, derived from mass deposition rates during application and monitoring of air. We estimated short and long-term malathion and malaoxon levels on outdoor surfaces, plants, and soils (0.1 and 1.0 cm mixing depth), using a simple first-order exponential decay model and literature half-life values ranging from three to nine days. Direct monitoring data were used to characterize short-term air levels. Average and upper-bound malathion levels immediately following an application were 0.091 microgram/m3 and 0.207 microgram/m3 in air; 22 and 52 mg/m2 on outdoor surfaces; 3.8 and 9.6 micrograms/g in plants; and 1.5 and 3.5 micrograms/g in soil (1 cm mixing depth). Malaoxon levels were 0.039 microgram/m3 and 0.110 microgram/m3 in air; 0.15 and 0.46 mg/m2, 0.03 and 0.09 microgram/g, and 0.01 and 0.03 microgram/g, respectively. Estimates of average and upper-bound levels over the duration of the eradication program were roughly one-third the immediate levels in all media. No field measurements were available for co-products other than malaoxon, for environmental persistence, or for concentrations on surfaces, soil, or plants after repeated applications. Because of limited monitoring data, and the likelihood that similar pest-eradication programs will be conducted, additional studies are warranted to more accurately characterize the environmental fate of malathion and human exposures after aerial application to residential communities.

Assessment of exposure to malathion and malaoxon due to aerial application over urban areas of southern California.

The state of California conducted aerial applications of malathion (MA) bait over urban areas in the southern California air basin in order to eradicate the Mediterranean Fruit Fly (Ceratitis capitata). Concern about the potential human health effects of this activity prompted a risk assessment conducted by the California Department of Health Services. Estimates of potential human exposures to MA and its primary breakdown product, malaoxon, (MO) are based on assumptions of daily human activities which influence the rate of contact with MA and MO. Several exposure scenarios, representing a range of activity levels from sedentary to very active, are used as surrogates for a variety of human activities. For each exposure scenario, acute dose rates are calculated using both the mean and the mean plus 2 standard deviations (SD) (98% upper confidence limit (UCL)) of measured environmental values. Chronic dose rates are calculated using long-term averages incorporating degradation and multiple applications at 14 day intervals, and the estimated 98% UCL for these averages. Based on this model, estimated adult dermal doses (1-246 micrograms/kg-d) are up to about 2000-fold higher than the estimated inhalation doses (0.01-0.1 microgram/kg-d) but are comparable to the doses from ingestion of contaminated unwashed backyard vegetables (30-80 micrograms/kg-d). For the individual who does not consume backyard vegetables, therefore, almost the whole dose of MA or MO would be due to contacting contaminated surfaces with skin.

ACTH regulation of cholesterol movement in isolated adrenal cells.

Confluent bovine adrenal cell primary cultures respond to stimulation by adrenocorticotropin (ACTH) to produce steroids (initially predominantly cortisol and corticosterone) at about one-tenth of the output of similarly stimulated rat adrenal cells. The early events of steroidogenesis, following ACTH stimulation, have been investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-6-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide (AMG), even though cytochrome P-450scc only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5-25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. More prolonged exposure to ACTH prior to addition of AMG caused the elevation in mitochondrial cholesterol to more than double, possibly due to enhanced binding capacity. Early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial-free cholesterol. The maximum rate of cholesterol transport to mitochondria in AMG-blocked cells was consistent with the maximum rate of cellular steroidogenesis. Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8) M), for 24 h prior to ACTH addition decreased the rates of ACTH- and cAMP-stimulated steroidogenesis but did not affect the basal rate. In both cases, the effectiveness of TCDD increased with time of exposure to the stimulant. Although cholesterol accumulated in the presence of ACTH and AMG (13-28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). Since even in the absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h) TCDD pretreatment may increase the presence of a protein(s) that cause this mitochondrial-cholesterol depletion following stimulation by ACTH or cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered regulation of adrenal steroidogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats.

A single treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (50 micrograms/kg) produced two distinct effects on adrenal steroidogenesis in rats 13 days post-treatment. In unstressed rats, the very low corticosterone levels early in the light phase (AM) increased 4-fold relative to ad libitum-fed control (ALC) rats, but the peak level of corticosterone that is seen late in the light phase (PM) decreased up to 40% relative to ALC rats. The AM stimulation was also observed in rats pair-fed to compensate for the diminished feed intake of TCDD-treated animals, indicating that the change results from nutritional deprivation. The PM suppression, however, was not observed in pair-fed rats. In rats given a lower dose of TCDD (15 micrograms/kg), there was no AM stimulation, whereas the suppression of the PM diurnal peak of corticosterone was retained. Plasma adrenocorticotropin (ACTH) levels and adrenal size were not changed by these treatments, indicating that TCDD affects adrenal responsiveness. TCDD did not, however, have a significant effect on corticosterone secretion in rats receiving high doses of ACTH. In control animals, the availability of cholesterol to cytochrome P-450scc limits the rate of steroidogenesis. While the specific content of the cytochrome was unaffected by TCDD, cholesterol turnover by this enzyme appeared to be affected following TCDD treatment, as evidenced by small increases in the mitochondrial levels of free cholesterol, reactive cholesterol, and in the proportion of P-450scc complexed with cholesterol relative to both ad libitum- and pair-fed controls. This accumulation of mitochondrial cholesterol following TCDD treatment is consistent with an inhibition of cholesterol metabolism at cytochrome P-450scc in vivo that is removed upon isolation of the mitochondria. These TCDD-induced increases were enhanced substantially in ACTH-stimulated rats, probably because ACTH enhances cholesterol influx into the mitochondria. Normally, substrate availability is rate limiting in cholesterol side-chain cleavage, and the AM stimulation of steroidogenesis by TCDD may result from such increased cholesterol transfer. The inhibition of cholesterol side-chain cleavage resulting from TCDD treatment may, however, only become rate limiting for corticosterone synthesis when cholesterol transfer is more substantially activated, as for peak PM secretion.

Hypercholesterolemia and the regulation of adrenal steroidogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats.

Plasma and adrenal cholesterol disposition have been examined to gain further insight into the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment decreases the diurnal peak in plasma corticosterone concentrations. TCDD induces an increase in plasma cholesterol concentration that is nearly complete on Day 2, at least 2 days before the most pronounced increase in adrenal cholesterol concentration (Days 4-6). This adrenal increase involves both free cholesterol and cholesterol esters, in contrast to the response to dietary hypercholesterolemia where only cholesterol esters increase. Although adrenocorticotropin (ACTH) does not increase adrenal mitochondrial cholesterol in normal rats (cholesterol turnover is faster than cholesterol uptake), this response changes between Days 6 and 9 after TCDD treatment such that ACTH then stimulates accumulation of mitochondrial cholesterol. This additional cholesterol is fully available to cytochrome P-450SCC, as judged both by active cholesterol metabolism in isolated mitochondria and by increased cholesterol-P-450SCC complex formation. The accompanying in vivo suppression of the peak plasma corticosterone concentration suggests a TCDD-induced inhibition of cholesterol side-chain cleavage (SCC). Consistent with this hypothesis, similar effects on adrenal mitochondrial cholesterol were produced by in vivo administration of the cholesterol side-chain cleavage inhibitor, aminoglutethimide, to ACTH-stimulated rats. Although the putative TCDD-induced inhibitory factor is apparently readily lost from mitochondria during preparation, inhibition may be retained in isolated cells. TCDD, therefore, affects adrenal cholesterol regulation by at least two mechanisms. Adrenal cholesterol content increases in part as a consequence of elevated plasma cholesterol, and cholesterol side-chain cleavage becomes partially inhibited in vivo.

Inhibition of ACTH action on cultured bovine adrenal cortical cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin through a redistribution of cholesterol.

The conversion of cholesterol to cortisol by cultured bovine adrenal cortical cells is stimulated 6-fold by adrenocorticotropin and is limited by the movement of cholesterol to the mitochondria (DiBartolomeis, M.J., and Jefcoate, C.R. (1984) J. Biol. Chem. 259, 10159-10167). Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8)M), for 24 h prior to adrenocorticotropin (ACTH) addition decreased the rate of ACTH-stimulated steroidogenesis but did not affect the basal rate. TCDD was more effective against stimulation at 10(-11) M ACTH (4-fold) than at 10(-7) M ACTH (10%), consistent with an increase in EC50 for ACTH. Stimulation of bovine adrenal cortical cells by cAMP was similarly decreased by TCDD. In both cases the effectiveness of TCDD increased with time of exposure to the stimulant. The transfer of cholesterol to mitochondria in intact cells was quantitated by means of the 2-h accumulation of mitochondrial cholesterol in the presence of aminoglutethimide, an inhibitor of cholesterol side chain cleavage. Although cholesterol accumulated in the presence of ACTH (13 to 28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13 to 8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). In absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h). It is concluded that TCDD pretreatment may increase the presence of a protein(s) that cause mitochondrial cholesterol depletion when the cells are stimulated by ACTH or cAMP. TCDD-enhanced cholesterol efflux from mitochondria diminishes cholesterol side chain cleavage when mitochondrial cholesterol is sufficiently depleted (after 2-4 h).

Regulation of rat and bovine adrenal metabolism of polycyclic aromatic hydrocarbons by adrenocorticotropin and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on polycyclic aromatic hydrocarbon (PAH) metabolism and steroidogenesis in primary cultures of bovine adrenal cortical (BAC) and rat adrenal cortical (RAC) cells have been examined. Remarkably TCDD is an ineffective inducer (15-50%) of PAH metabolism in confluent BAC cells and completely antagonizes a 5-fold induction by benz[alpha]anthracene (BA). In the same concentration range (EC50 5 X 10(-11) M) TCDD suppresses steroidogenesis through an effect on cholesterol metabolism. Adrenocorticotropin (ACTH) and cAMP also suppress PAH metabolism at concentrations which stimulate steroidogenesis (10(-7) M). In RAC cells ACTH potently induces PAH metabolism (7-fold) at a comparable concentration to the stimulation of steroidogenesis. Parallel stimulation of PAH metabolism and steroidogenesis by cAMP suggest that ACTH induction of PAH metabolism is mediated by cAMP. TCDD induces PAH metabolism (2.8-fold, EC50 8 X 10(-11) M) at similar concentrations to the inhibitory effect in BAC cells and this action is additive with ACTH induction. In male rats in vivo TCDD induces adrenal microsomal PAH metabolism (72%) and is more effective in this respect than 3-methylcholanthrene (3MC). Rabbit antibodies against rat liver cytochrome P-450c (the major TCDD-inducible liver form) inhibited the TCDD-induced adrenal metabolism of 7,12-dimethylbenz[alpha]anthracene (DMBA), which also exhibited regioselectivity typical of metabolism by P-450c. Constitutive adrenal microsomal metabolism, which exhibited regioselectivity of DMBA metabolism comparable to the ACTH-sensitive cellular metabolism, was not affected by anti-P-450c. It is concluded that ACTH and TCDD induce distinct forms of cytochrome P-450 in RAC cells and that the latter represents a typical Ah-receptor mediated response. The anomalous effect on PAH metabolism in BAC cells that parallels inhibition of steroidogenesis may derive from repression of a distinct adrenal form of P-450 by the TCDD-Ah-receptor complex.

Control of steroid synthesis in adrenal fasciculata cells.

The control of steroid synthesis in adrenal fasciculata cells is considered in terms of two types of control by ACTH: control of cholesterol availability to inner mitochondrial cytochrome P-450scc. This process controls total steroid synthesis and is rapidly activated by ACTH. The several steps in cholesterol transfer are examined. partitioning of metabolism by means of competition between enzymes for limiting amounts of steroid intermediates. Changes in such competition determine the ratio of steroid products from the adrenal cells. Such changes typically are a slower response to ACTH. A critical aspect of such competition is the modulation of multiple activity P-450 cytochromes: P-450(17 alpha) (17 alpha-hydroxylation and 17,20 lyase) and P-450(11 beta) (11 beta- and 18-oxidases). Factors such as substrate binding, electron transfer steps and lipid environment are considered in addition to new enzyme synthesis. The ACTH stimulation of steroid synthesis in bovine adrenal cell primary cultures is examined as a model for both types of regulation.

Characterization of the acute stimulation of steroidogenesis in primary bovine adrenal cortical cell cultures.

The early events of steroidogenesis, following adrenocorticotropin (ACTH) stimulation, were investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide, even though cytochrome P-450scc only increases after 12 h. Mitochondrial free cholesterol levels increased during the same time period (16.5 to 25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. It is concluded that early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial free cholesterol. The maximum rate of cholesterol transport to mitochondria in aminoglutethimide-blocked cells (8.6 micrograms/mg of protein/h) was consistent with both the maximum rate of mitochondrial cholesterol SCC and cellular steroidogenesis (6.0 micrograms of pregnenolone/mg/h and 5.5 micrograms of steroid/mg of mitochondria/h, respectively). Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial free cholesterol. The distribution of steroid products fell into three distinct phases during a 24-h period following ACTH stimulation: an initial increase in SCC activity (0-4 h), elevation of androstenedione in place of corticosterone (4-12 h), and then in place of cortisol (12-24 h). The changes from 4 to 24 h result from a progressive stimulation by ACTH of 17 alpha-hydroxylase activity (but not 21-hydroxylase or C17:20 lyase activities) that is maintained even when stimulation of total steroidogenesis has stopped.

The interrelationship of polycyclic hydrocarbon metabolism and steroidogenesis in primary cultures of bovine adrenal cortical cells.

The interrelationship between adrenal steroidogenesis and polycyclic aromatic hydrocarbon metabolism has been examined in cultured bovine adrenal cortical (BAC) cells. Adrenocorticotropin (ACTH) selectively induced steroidogenic cytochrome P-450-dependent enzyme activities from BAC cell cultures. In the presence of 10(-7) M ACTH, steroid production requiring 17 alpha-hydroxylation (cortisol + androgens) was increased 5-fold over the formation of 17- deoxysteroids (corticosterone). The effect of 10 microns benz[a]anthracene on steroidogenesis was characterized by suppression of both steroid 17 alpha-hydroxylation (90%) and total steroidogenesis (50%), with a concomitant rise in 17- deoxysteroid formation. The order of stimulation of steroidogenic enzyme activities by ACTH (17 alpha-hydroxylase greater than side chain cleavage greater than 21-hydroxylase) paralleled the order of suppression by benz[a]anthracene. BAC cell cultures incubated with Su-10603, a specific 17 alpha-hydroxylase inhibitor, exhibited similar changes in the pattern of steroidogenesis, as did benz[a]anthracene-treated cells, suggesting that benz[a] anthracene also inhibits steroidogenesis as an inhibitor of 17 alpha-hydroxylase. In addition, benz[a]anthracene induced benzo[a]pyrene metabolism 4- to 6-fold over control levels in these cells. The profile of benzo[a]pyrene metabolites revealed predominantly water-soluble products (nonhydrolyzable greater than sulfates greater than glucuronides), 9,10- monooxygenation products, and 3-phenol. ACTH (10(-7) M) and 0.5 mM cyclic AMP each decreased benzo[a]pyrene metabolism by more than 50%. Both benz[a]anthracene-induced and uninduced benzo[a]- pyrene metabolism were equally reduced in response to ACTH and cyclic AMP. In the presence of 0.2 mM aminoglutethimide, which completely inhibited steroidogenesis, ACTH decreased benz[a]anthracene induction of benzo[a]pyrene metabolism to the same extent as ACTH treatment alone. It is concluded that the suppression of benzo[a]pyrene metabolism by ACTH is mediated by cyclic AMP and does not involve steroids generated in response to ACTH. These studies demonstrate that cytochrome P-450 isozymes involved in steroidogenesis and polycyclic aromatic hydrocarbon metabolism are regulated, in opposing directions, by ACTH.

Isolation and development of chlorosomes in the green bacterium Chloroflexus aurantiacus.

Freeze-fracture electron microscopy was used to study further the changes in chlorosome structure during the development of the photosynthetic apparatus in Chloroflexus aurantiacus J-10-fl. During development, in response to decreased light intensity or lower oxygen tension, the number of chlorosomes per cell increased. The same conditions also led to a general thickening of chlorosomes but did not affect their length or width. The thickening of the chlorosomes paralleled increases in the bacteriochlorophyll c/bacteriochlorophyll a ratio. Semiaerobic induction of the photosynthetic apparatus did not produce a synchronous assembly of chlorosomes in all cells of a given culture. Even adjacent cells of a single filament showed great variations in the rate and extent of response. Parallel appearance of (i) approximately 5-nm particles (in a lattice configuration) in the membrane attachment site, (ii) the crystalline baseplate material (with a periodicity of approximately 6 nm) adjacent to the membrane attachment site, and (iii) the chlorosome envelope layer preceded addition of longitudinally oriented, rodlike elements (diameter, congruent to 6 m) to the chlorosome core. It is estimated that each chlorosome can funnel energy into approximately 100 reaction centers. Chlorosomes could be isolated by a simple density gradient procedure only from cells grown at low light intensity. A bacteriochlorophyll a species absorbing at 790 nm was associated with isolated chlorosomes. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorosomes showed only a few low-molecular-weight polypeptides (less than 15,000).

A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2).

In previous work we have demonstrated that the microtubule-associated protein 2 (MAP 2) molecule consists of two structural parts. One part of the molecule, referred to as the assembly-promoting domain, binds to the microtubule surface and is responsible for promoting microtubule assembly; the other represents a filamentous projection observed on the microtubule surface that may be involved in the interaction of microtubules with other cellular structures. MAP 2 is known to be specifically phosphorylated as the result of a protein kinase activity that is present in microtubule preparations. We have now found that the activity copurifies with the projection portion of MAP 2 itself. Kinase activity coeluted with MAP 2 when microtubule protein was subjected to either gel- filtration chromatography on bio-gel A-15m or ion-exchange chromatography on DEAE- Sephadex. The activity was released from microtubules by mild digestion with chymotrypsin in parallel with the removal by the protease of the MAP 2 projections from the microtubule surface. The association of the activity with the projection was demonstrated directly by gel filtration chromatography of the projections on bio-gel A-15m. Three protein species (M(r) = 39,000, 55,000, and 70,000) cofractionated with MAP 2, and two of these (M(r) = 39,000 and 55,000) may represent the subunits of an associated cyclic AMP- dependent protein kinase. The projection-associated activity was stimulated 10-fold by cyclic AMP and was inhibited more than 95 percent by the cyclic AMP-dependent protein kinase inhibitor from rabbit skeletal muscle. It appeared to represent the only significant activity associated with microtubules, almost no activity being found with tubulin, other MAPs, or the assembly-promoting domain of MAP 2, and was estimated to account for 7-22 percent of the total brain cytosolic protein kinase activity. The location of the kinase on the projection is consistent with a role in regulating the function of the projection, though other roles for the enzyme are also possible.