Atherosclerotic cardiovascular disease screening and management protocols among adult HIV clinics in Asia.

OBJECTIVES: Integration of HIV and non-communicable disease services improves the quality and efficiency of care in low- and middle-income countries (LMICs). We aimed to describe current practices for the screening and management of atherosclerotic cardiovascular disease (ASCVD) among adult HIV clinics in Asia. METHODS: Sixteen LMIC sites included in the International Epidemiology Databases to Evaluate AIDS - Asia-Pacific network were surveyed. RESULTS: Sites were mostly (81%) based in urban public referral hospitals. Half had protocols to assess tobacco and alcohol use. Protocols for assessing physical inactivity and obesity were in place at 31% and 38% of sites, respectively. Most sites provided educational material on ASCVD risk factors (between 56% and 75% depending on risk factors). A total of 94% reported performing routine screening for hypertension, 100% for hyperlipidaemia and 88% for diabetes. Routine ASCVD risk assessment was reported by 94% of sites. Protocols for the management of hypertension, hyperlipidaemia, diabetes, high ASCVD risk and chronic ischaemic stroke were in place at 50%, 69%, 56%, 19% and 38% of sites, respectively. Blood pressure monitoring was free for patients at 69% of sites; however, most required patients to pay some or all the costs for other ASCVD-related procedures. Medications available in the clinic or within the same facility included angiotensin-converting enzyme inhibitors (81%), statins (94%) and sulphonylureas (94%). CONCLUSION: The consistent availability of clinical screening, diagnostic testing and procedures and the availability of ASCVD medications in the Asian LMIC clinics surveyed are strengths that should be leveraged to improve the implementation of cardiovascular care protocols.

Genotoxicity profiles of common alkyl halides and esters with alkylating activity.

Drug synthesis and/or formulation can generate genotoxic impurities. For instance, strong acid/alcohol interactions during the process of drug salt formation produce alkylating agents such as alkyl halides and alkyl esters of alkyl sulfonic acids. The genotoxicity of a few classic alkylating agents such as methyl and ethyl methanesulfonate have been previously well characterized, whereas the majority of compounds from this class have only been tested in the Salmonella reversion assay. Therefore, the goal of this study was to investigate clastogenicity and DEL recombination profiles of 22 halogenated alkanes and alkylesters of sulfuric and alkane-, aryl-sulfonic acids using a battery of cellular and molecular assays. The in-vitro micronucleus assay in CHO cells was used to measure clastogenicity and the deletion recombination (DEL) assay in S. cerevisiae provided a measure of DNA deletions. We also examined the compounds' reactivity towards 4-(p-nitrobenzyl)pyridine (NBP), a surrogate molecule for biological ring nitrogens. Methylating agents were most potent in all three assays and the alkyl chlorides evaluated in our study were negative in all three assays. Also, a strong correlation was found between the MN, DEL and NBP assays. In summary, this study contributes to a better understanding of the genotoxic properties of common alkyl halides and alkyl esters with alkylating activity and might provide guidance for managing risk of genotoxic process-related impurities of drug substances and products.

Lanosterol 14alpha-demethylase (CYP51), NADPH-cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol.

Lanosterol 14alpha-demethylase (CYP51) is a cytochrome P450 enzyme involved primarily in cholesterol biosynthesis. CYP51 in the presence of NADPH-cytochrome P450 reductase converts lanosterol to follicular fluid meiosis activating sterol (FF-MAS), an intermediate of cholesterol biosynthesis which accumulates in gonads and has an additional function as oocyte meiosis-activating substance. This work shows for the first time that cholesterogenic enzymes are highly expressed only in distinct stages of spermatogenesis. CYP51, NADPH-P450 reductase (the electron transferring enzyme needed for CYP51 activity) and squalene synthase (an enzyme preceding CYP51 in the pathway) proteins have been studied. CYP51 was detected in step 3-19 spermatids, with large amounts in the cytoplasm/residual bodies of step 19 spermatids, where P450 reductase was also observed. Squalene synthase was immunodetected in step 2-15 spermatids of the rat, indicating that squalene synthase and CYP51 proteins are not equally expressed in same stages of spermatogenesis. Discordant expression of cholesterogenic genes may be a more general mechanism leading to transient accumulation of pathway intermediates in spermatogenesis. This study provides the first evidence that step 19 spermatids and residual bodies of the rat testis have the capacity to produce MAS sterols in situ.

The effects of dietary boric acid on bone strength in rats.

The effects of dietary boron (B) (from boric acid [BA]) on bone strength were evaluated using male F344 rats. B was administered by dietary admixture of BA to NIH-07 feed at concentrations of 200, 1000, 3000, and 9000 ppm. The latter two levels were found in previous studies to be reproductively toxic to both males and the developing fetus. The first two levels are below and just at, respectively, the levels for producing fetal malformations, and are below the dose required to produce male reproductive toxicity. Resistance to destructive testing was measured on femora, tibiae, and lumbar vertebrae. Although femur and tibia resistance to bending force were not affected by any amount of dietary B, vertebral resistance to a crushing force was increased by approximately 10%, at all dose levels (200-9000 ppm). These data show that even levels of BA that are not reproductively toxic can affect the strength of the axial skeleton in rats.

Cyclophilin A is present in rat germ cells and is associated with spermatocyte apoptosis. Reproductive Toxicology Group.

Recent investigations in our laboratory revealed divalent cation-dependent endonuclease activity in testes from 2-methoxyethanol-treated rats, which was able to cleave substrate DNA into a pattern of DNA fragmentation consisting of approximately 180-200 base pairs. Further studies were undertaken to characterize the active nuclease. F344 rats were treated with 2-methoxyethanol, a glycol ether that causes the death of pachytene spermatocytes in juvenile and adult rats. The active nuclease was found in nuclear extract from treated animals, but not controls. A radioactive gel nuclease assay, which detects degradation and loss of 32P-labeled DNA from a DNA-containing polyacrylamide gel, localized the nuclease activity to a band of approximately 18 kDa. This activity was dependent on calcium and was inhibited by both zinc and aurintricarboxylic acid. Amino acid sequence data showed that this protein was identical to cyclophilin A. Immunohistochemistry using antibodies against cyclophilin A found specific staining in pachytene spermatocytes, spermatids, interstitial cells, and Sertoli cell nuclei. Cyclophilin A staining was present in both control and 2-methoxyethanol-treated rat testes in a stage-dependent manner, with pachytene spermatocytes in stage-VIII-XIV seminiferous tubules most heavily stained. These data demonstrate that rat testis germ cells contain relatively high levels of cyclophilin A whose nuclease activity is associated with spermatocyte apoptosis induced by 2-methoxyethanol.

The effects of dietary boron on bone strength in rats.

Previous studies from our laboratory found that when boric acid (BA) was administered in the diet to rats, boron levels in bone were approximately fourfold greater than serum levels. The current studies were undertaken to determine if these elevations produced adverse effects on several bone-related measures, including serum electrolyte levels, bone structure, and bone strength. Data from two studies are presented: in the first study, young adult male rats consumed a powdered diet containing 0, 3000, 4500, 6000, or 9000 ppm BA for 9 weeks. Endpoints were serum calcium, phosphorous, potassium, and chloride, as well as blood and bone boron concentrations ([B]) measured weekly during the 9-week exposure period, and at 8, 16, 24, and 32 weeks after the end of exposure. In the second study, the male and female young adult rats diet contained 0, 200, 1000, 3000, or 9000 ppm BA for 12 weeks; endpoints measured weekly were serum levels of calcium, phosphorous, and magnesium, bone [B], and bone structure (humerus) and strength (tibia, femur, and lumbar vertebrae). In treated rats, calcium was reduced in the first study but not the second. Serum phosphorous was reduced in both studies; potassium was unchanged, chloride was increased by 1%, and magnesium was reduced in all BA-exposed groups in the second study, to a maximal 19% reduction. Bone [B] was consistently increased in all treated groups, to concentrations approximately fourfold those of serum. After cessation of exposure, serum and urinary boron concentrations dropped to within control values within a week. However, even 32 weeks after the end of exposure, bone [B] remained threefold greater than controls. Male tibia and femur resistance to bending was unchanged. However, vertebral strength in compression was significantly increased by 5-10% in all dose groups (200 to 9000 ppm). The pattern was substantially similar in females. Only the humerus was examined by light microscopy and was found to be unchanged at any level of BA consumption. These data show that, despite a reduction in some serum electrolyte levels, BA consumption increased vertebral resistance to crush force, without detectably altering the microscopic structure of the humerus or the resistance of femur and tibia to a bending load. This increase in compression resistance occurred at exposure levels substantially below those that were previously reported to be reproductively toxic.

Spermatocyte toxicity of 2-methoxyethanol (ME) in rats and guinea pigs: evidence for the induction of apoptosis.

2-Methoxyethanol (ME) produces testicular lesions characterized by pachytene spermatocyte degeneration in rats and guinea pigs which differ in onset, severity, and morphological characteristics. In the rat, degenerating spermatocytes appear necrotic at 24 hr, while in the guinea pig they appear apoptotic 96 hr after the start of three daily doses. To further examine if the spermatocyte degeneration in both species represented necrosis or apoptosis, the extent and nature of nuclear DNA fragmentation after ME exposure were assessed both visually using an in situ nucleotide 3' end-labeling (ISEL) procedure and by DNA gel electrophoresis. Testes from rats given a single oral dose of ME (200 mg/kg) showed the expected pachytene spermatocyte degeneration 24 hr after dosing, with the nuclear chromatin degradation typical of necrosis. In contrast, testes from guinea pigs given daily oral doses of ME (200 mg/kg) showed spermatocyte degeneration at only 96 hr after the start of dosing, with marked peripheral nuclear chromatin condensation characteristic of apoptosis. Coincident with the appearance of morphologic changes, degenerating spermatocytes in both species contained fragmented DNA as revealed by the ISEL procedure. The pattern of DNA fragmentation on agarose gels in both species consisted of ordered multiples or "ladders" of approximately 200 base pairs, a hallmark of apoptosis, with their appearance coincident with the time course of morphologic spermatocyte degeneration and ISEL staining. Preliminary data reveal the appearance of divalent metal cation-dependent endonuclease activity at pH 7.0 in ME-treated immature (24-day-old) rat testis that produces a similar pattern of DNA fragmentation and which appears to be distinct from activity associated with the spontaneous germ cell degeneration observed in testes of this age. In summary, in vivo ME exposure induces spermatocyte apoptosis in both the rat and guinea pig despite differing morphological classifications and time of onset of cell death. Future studies will focus on further characterization of the testicular endonuclease in the rat and the potential role of increased intracellular Ca2+ as a "triggering" stimulus in ME-induced spermatocyte apoptosis.

Histologic localization of surgically transplanted syngeneic seminiferous tubule segments into testis with Fast Blue as tracer.

Spermatogenesis is a complex differentiative process influenced by the testicular extratubular and intratubular tissue environments. One method of determining the relative importance of intratubular versus extratubular factors in cases of deficient spermatogenesis has been syngeneic seminiferous tubule transplantation. Generally in such a scheme, tubule segments from a testis deficient in spermatogenesis are transplanted into an intact recipient testis, and the progression of spermatogenesis in transplanted tubules is examined histologically. However, this experimental approach has been complicated by the tedious histologic serial sectioning required to locate these transplanted tubules and the need to distinguish them from recipient testis solely by structural differences. A method is described for the surgical transplantation of seminiferous tubule segments into rat testes that uses prelabeling donor tubules in vitro with the fluorescent tracer Fast Blue to facilitate their localization. The technique was evaluated by transplanting cut segments of Fast Blue-labeled seminiferous tubules from 15-day-old rat testis into normal adult rat testis (recipient), then localizing and histologically examining the progression of spermatogenesis in the transplanted tubules for up to 28 days. Transplanted tubules were easily identified in sections of recipient testis by fluorescent microscopy; intense Fast Blue staining with low background was seen up to 28 days after transplantation. Histologically, transplanted tubules had limited germ cell differentiation in recipient testis for the Fischer rat strain. At 10 days after transplantation, tubules had characteristics qualitatively similar to tubules from 25-day-old rat testis, with increased tubular diameter and abundant germ cells at the pachytene spermatocyte stage.(ABSTRACT TRUNCATED AT 250 WORDS)

The reproductive toxicity of boric acid.

Previous studies on the reproductive toxicity of boric acid have indicated that male rodents suffer testicular atrophy after treatment. There were, however, no studies of the potential effects on female fertility or on the neonate. In addition, no study described the development of the testicular lesion, thought to be related to the mechanism of toxicity. A Reproductive Assessment by Continuous Breeding (RACB) study using mice exposed to boric acid at 1000, 4500, and 9000 ppm in the diet indicated that there are probably multiple sites of action, although male fertility appears very sensitive. Possible effects on female fertility cannot be separated from potential developmental toxicity and need additional investigation. Decrements in sperm motility were observed at all exposure levels, and testicular atrophy was confirmed in high- and middle-dose-group males. This was investigated further by timed serial-sacrifice studies using 9000 ppm in the diet of rats, which found that the first lesion seen in the testis was an inhibition of spermiation (release of mature spermatids). With continued dosing, this was followed by a disorganization of the normal ordered layering of the seminiferous epithelium, germ cell sloughing and death, and finally, atrophy. Subsequent studies using additional doses (2000, 3000, 4500, 6000, and 9000 ppm) found that it was possible to observe inhibited spermiation that did not progress to atrophy (4500 ppm and below) within the 9-week exposure period.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the testicular toxicity of boric acid in rats: in vivo and in vitro studies.

High-dose boric acid (BA) exposure produces testicular lesions in adult rats characterized by inhibited spermiation (IS) that may progress to atrophy. In vivo and in vitro studies addressed possible mechanisms. In vivo, boron tissue disposition was examined, since no detailed data existed, and relevant boron concentrations for in vitro studies needed to be set. Since BA induces riboflavinuria and also affects calcium/phosphorus homeostasis, and testis zinc appears essential for normal testis function, we examined BA effects on flavin status and testis levels of phosphorus (P), calcium (Ca) and zinc (Zn). Data showed that the testicular toxicity and central nervous system (CNS) hormonal effect were not due to selective boron accumulation in testis or brain/hypothalamus, with testis boron concentrations at approximately 1 to 2 mM; that riboflavin deficiency is not involved, due to both the absence of overt signs of deficiency and effects on tissue flavin content during BA exposure; and that changes in testis P, Ca and Zn levels did not precede atrophy, and are therefore unlikely to be mechanistically relevant. In vitro studies addressed the hallmarks of the BA testicular toxicity: the mild hormone effect, the initial IS, and atrophy. No effect of BA on the steroidogenic function of isolated Leydig cells was observed, supporting the contention of a CNS-mediated rather than a direct hormone effect. Since increased testicular cyclic adenosine monophosphate (cAMP) produces IS, and a role for the serine proteases plasminogen activators (PAs) in spermiation has been proposed, we examined in vitro BA effects on both Sertoli cell cAMP accumulation and PA activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the testicular effects of 2-methoxyethanol (ME) in rats and guinea pigs.

Glycol ethers produce both hemato- and testicular toxicity in animals, which is dependent on both the alkyl chain length and animal species used. Ethylene glycol monobutyl ether (2-butoxyethanol, BE) causes hemolytic anemia in rats but not in guinea pigs, and red blood cells from both guinea pigs and humans are minimally affected in vitro by the active metabolite 2-butoxyacetic acid. This demonstrates the importance of animal species selection for assessing human risk to BE exposure. 2-Methoxyethanol (ME) produces testicular lesions in rats characterized primarily by the degeneration of spermatocytes undergoing meiotic division with minimal or no hemolytic changes. Because of the differential hemolytic response to BE between rats and guinea pigs, the present study addressed whether the testicular response to ME was similarly dichotomous. Adult rats or guinea pigs were given a single dose of either 200 or 300 mg ME/kg by gavage, and testicular and hemolytic changes were assessed 24 hr after treatment. Testis histology in rats showed dose-dependent degeneration of dividing spermatocytes in stage XIV tubules as expected, with only minimal hemolytic changes, also as expected. In contrast, no testicular or hemolytic effects were observed in guinea pigs 24 hr after either single ME dose. In a subsequent study, a single dose or multiple (3 daily) doses of 200 mg ME/kg were given, and animals were examined at 4 days after the start of treatment. Testes from rats given both single and multiple ME doses showed, as expected, tubules depleted of spermatocytes and early spermatids. In guinea pigs, spermatocyte degeneration was observed in stage III/IV tubules for both dosing schemes, but was much less severe and widespread and differed from rats in morphological characteristics, specifically in the appearance of nuclear chromatin degeneration. In the rat, degenerating spermatocytes showed uniformly condensed and dispersed chromatin, while in the guinea pig they showed marked chromatin condensation at the nuclear periphery. No hemolytic changes were observed in either species or dosing scheme. In summary, although ME-associated testicular lesions were observed in both species, they differed significantly in onset, characteristics, and severity. Both the nature of the differential testicular response to ME and a comparison to the in vitro human testicular response to the active metabolite 2-methoxyacetic acid are subjects of future study.

Spermatocyte toxicity of 2-methoxyethanol in vivo and in vitro: Requirement for an intact seminiferous tubule structure for germ cell degeneration.

Cultures of isolated testicular cells are widely used for evaluating mechanisms of action of direct-acting testicular toxicants. However, for testicular cells, the expression of toxicity in vitro is frequently different from that found in vivo. 2-Methoxyethanol (ME) produces testicular lesions in rats which are characterized by pachytene spermatocyte degeneration 24 hr after dosing. As part of a study to evaluate mechanisms of male germ cell toxicants, we compared the morphological aspects of spermatocyte toxicity after in vivo exposure to ME with those found in various testicular cell culture systems after in vitro exposure to the active ME metabolite, 2-methoxyacetic acid (MAA). Immature rats were used because they respond to in vivo ME treatment in the same way as adults, but their testes are relatively enriched in pachytene spermatocytes. 24-day-old rats were given a single dose of 250 mg ME/kg body weight by gavage and the testes were evaluated 24 hr after dosing. In vitro, cultured seminiferous tubules, Sertoli-germ cell co-cultures, and enriched mixed germ cells, all prepared from 24-day-old rats, were evaluated after 24-hr in vitro exposure to 5 mm MAA (a level of MAA found after ME exposure in vivo). Testes from ME-exposed rats showed the expected pachytene spermatocyte degeneration 24 hr after dosing. Similar changes were observed in cultured seminiferous tubules after 24 hr of exposure to MAA in vitro. However, without the intact seminiferous tubule structure in vitro, the expression of MAA spermatocyte toxicity was different. In conventional Sertoli-germ cell co-cultures, spermatocyte detachment from the Sertoli cell monolayer occurred as expected, although no significant morphological degeneration was observed in these detached germ cells. Similarly, no increase in degenerating spermatocytes was noted in isolated enriched mixed germ cells after in vitro MAA exposure. Instead, toxicity in these germ cell fractions was expressed only by increased uptake of trypan blue dye, revealing an increase in plasma membrane permeability. In summary, this in vivo/in vitro comparison of the spermatocyte toxicity of ME/MAA showed that the expression of toxicity is different in the different culture architectures and that an intact seminiferous tubule structure is required for full expression of the morphological degeneration produced by ME/MAA, and suggests the usefulness of culture seminiferous tubules in future mechanistic studies.

The effects of boric acid (BA) on testicular cells in culture.

High-dose boric acid (BA) exposure produces testicular lesions in adult rats characterized by inhibited spermiation that may progress to nonrecoverable atrophy. The mechanism for the testicular toxicity of BA is unknown. To examine possible direct effects, the present study evaluated selected aspects of various testicular cell culture systems after in vitro BA exposure. Specifically, the hallmarks of the BA testicular toxicity were addressed: the mild hormone effect, the initial inhibition of spermiation, and atrophy. No effect of BA on the steroidogenic function of isolated Leydig cells was observed, supporting the contention of a CNS-mediated rather than a direct hormone effect. Since increased testicular cyclic AMP (cAMP) produces inhibited spermiation, and a role for the serine proteases plasminogen activators (PAs) in spermiation has been proposed, we evaluated both Sertoli cell cAMP accumulation in Sertoli-germ cell cocultures and the stage-specific secretion of PA activity in cultured seminiferous tubules after in vitro BA exposure, respectively. The results showed that the inhibited spermiation is not due to BA effects on either process. To address the atrophy, we evaluated BA effects in Sertoli-germ cell cocultures on 1) morphology/germ cell attachment, which might identify a target cell; 2) Sertoli cell energy metabolism, because lactate, secreted by Sertoli cells, is a preferred energy source for germ cells; and 3) DNA/RNA synthesis, because germ cells synthesize DNA/RNA and BA impairs nucleic acid synthesis in liver and may do so in testis. Despite the absence of overt morphologic changes and germ cell loss, the most sensitive in vitro endpoint was DNA synthesis of mitotic/meiotic germ cells, with energy metabolism in Sertoli or germ cells affected to a lesser extent. A re-evaluation of testis sections from rats exposed to BA revealed a decrease in the early germ cell/Sertoli cell ratio prior to atrophy. Thus, although the mechanism for the inhibited spermiation is still undefined and is the subject of future work, these combined studies revealed some changes offering a plausible explanation for the atrophy aspect of the BA testicular lesion.

Testicular toxicity of boric acid (BA): relationship of dose to lesion development and recovery in the F344 rat.

High-dose boric acid (BA) produces testicular lesions in adult rats, characterized by inhibited spermiation followed by atrophy. The present study addressed whether inhibited spermiation can be separated from atrophy based on dose, compared testis boron (B) dosimetry to lesion development, determined how inhibited spermiation was reflected by common reproductive endpoints, and examined reversibility of the testicular lesions. Rats were fed 3000, 4500, 6000, or 9000 ppm BA for up to 9 weeks and examined. Recovery was assessed for up to 32 weeks post treatment. Inhibited spermiation could be separated from atrophy based on dose (inhibited spermiation: 3000/4500 ppm; atrophy: 6000/9000 ppm), with each lesion aspect expressed at different threshold testis B concentrations (inhibited spermiation: 5.6 micrograms B/g and atrophy: 11.9 micrograms B/g) with no B accumulation during the 9-week exposure. These data suggest that separate mechanisms may be operating for these lesion aspects based on testis B concentration and that B dose rate was important for testicular toxicity. Inhibited spermiation was most reliably reflected by informed testicular histology, with the more severe cases decreasing epididymal sperm count to levels that could affect fertility. After treatment, serum and testis B levels in all dose groups rapidly fell to background levels at the earliest time points evaluated (7 days and 8 weeks posttreatment, respectively). The severely inhibited spermiation at 4500 ppm was resolved by 16 weeks posttreatment, but areas of focal atrophy were detected that did not recover posttreatment. Also, no signs of recovery from atrophy were observed (6000 and 9000 ppm). Atrophic tubules contained a normal complement of spermatogonia (2.6 to 2.9 germ cells/100 Sertoli cells), with occasional dividing and degenerating germ cells. Elevations in serum FSH and LH levels suggested an intact hormonal response to the atrophy. In summary, 1) the different aspects of the BA-induced testicular lesion can be separated using different doses, 2) inhibited spermiation does not necessarily proceed to atrophy, and 3) there is no recovery from the atrophy despite the absence of testis B after treatment. The ability to separate inhibited spermiation from atrophy based on dose and testis B dosimetry will be useful in evaluating possible mechanisms. Furthermore, the presence of dividing spermatogonia during long-term BA-induced atrophy suggests that this model should be useful for identifying critical components involved in the reinitiation of spermatogenesis.

Tissue disposition of boron in male Fischer rats.

Boric acid (H3BO3), an inorganic acid with widespread commercial use and consumer exposure, impairs fertility in male rodents at dose levels lower than those required to cause other adverse effects. Previous studies found a testicular lesion in adult Fischer rats fed 9000 ppm boric acid (1575 ppm boron) and slightly reduced basal serum testosterone levels. A CNS-mediated hormonal component to this lesion was suggested. Detailed data on the tissue disposition of boron in the rat, including accessory sex organs and the brain, are lacking. This study examined the tissue disposition of boron in reproductive, accessory sex organs, and other selected tissues in adult male Fischer rats fed 9000 ppm boric acid to determine if selective accumulation of boron in reproductive tissues, accessory sex organs, and/or the brain might correlate with and explain the apparent selective testicular toxicity. Adult male Fischer rats were fed 9000 ppm boric acid for up to 7 days. Animals were killed at 1, 2, 3, 4, and 7 days after the start of exposure. Plasma and excised tissues were heat-digested in acid and analyzed for boron by inductively coupled argon plasma emission spectrometry (ICAP). With the exception of adrenal glands, control boron levels in all tissues examined were below 4 micrograms/g. There was a rapid increase in plasma and tissue boron 1 day after the start of exposure (range 2- to 20-fold), with the exception of adipose tissue. With the exception of bone and adipose tissue, all soft tissues examined, including the testis, epididymis, accessory sex organs, hypothalamus, and rest of brain, appeared to reach steady-state boron levels (range 12-30 micrograms/g) by 3-4 days. Bone boron levels continued to increase up to the termination at 7 days (40-50 micrograms/g by Day 7). Bone attained the greatest concentration of boron (2- to 3-fold over plasma levels) while levels in adipose tissue were 20% of plasma levels during the 7-day exposure period. All other tissues appeared to show no appreciable accumulation of boron over plasma levels. The data suggest that neither the apparent selective testicular toxicity nor the slight CNS hormonal effect associated with boric acid exposure can be explained on the basis of selective accumulation of boron in the testis or brain/hypothalamus, respectively. Thus, the testicular toxicity is likely the result of certain biological processes that are unique to the testis and which are targets of boron exposure.

Pteridine modulation of lead inhibition of uroporphyrinogen synthesis in erythroid precursor cells.

The role of nutritional factors on heme synthesis and their influence on the development of anemia in the bone marrow during lead exposure is unclear. Previous reports suggested that pteridines could regulate the formation of uroporphyrinogen, a step midway along the heme synthetic pathway. Studies were undertaken to determine if pteridines could modulate lead inhibition of uroporphyrinogen synthesis in erythroid precursor cells. Pteroylpolyglutamates of various glutamate chain lengths were tested for the ability to protect against lead inhibition of uroporphyrinogen I (URO) synthase prepared from murine erythroleukemia cells (MELC). Pteroylpentaglutamate, the major endogenous polyglutamate form by chain length found to be present in MELC, afforded rapid and specific protection of URO synthase against lead inhibition. MELC are expected to be a useful in vitro model for studying the role of endogenous folates on uroporphyrinogen synthesis and heme formation in erythroid precursor cells following lead exposure.

Effects of lead on haem biosynthesis during erythroid differentiation in vitro.

Murine erythroleukaemia cells (MELC) are erythroid precursor cells that undergo erythroid differentiation in the presence of the inducer hexamethylene bisacetamide (HMBA). The effects of lead on haem biosynthesis in MELC following HMBA-induced differentiation were studied. MELC were induced with HMBA in the presence of 20, 40 and 80 mum-lead acetate and cell density, haem content, incorporation of (14)C-labelled delta-aminolaevulinic acid (ALA) into haem, and the activities of the enzymes delta-aminolaevulinic acid dehydratase (ALA-D), uroporphyrinogen I synthetase (URO-S) and ferrochelatase (FERRO) were determined. MELC exposed to 80 mum-lead showed significant erythroid hypoplasia (40-50%) and a significant decrease (30-50%) in haem content at 2, 4 and 6 days after induction in comparison with the controls. Significant inhibition of ALA-D, the most sensitive index, was noted at 20 mum-lead, and at 80 mum-lead ALA-D activity was decreased by 60-80% in comparison with the controls. URO-S and FERRO showed significant decreases of 34% and 50%, respectively, at 80 mum-lead. A decrease of 50% in the incorporation of [(14)C]ALA into haem at 80 mum-lead indicated an impairment in haem synthesis. The results suggest that the impairment of haem formation by lead is coincident with the production of severe erythroid hypoplasia.

bis-(beta-chloroethyl)sulfide (BCES)-induced changes in epidermal cell homeostasis in vitro.

A rat cutaneous keratinocyte culture system was developed to study the effects of the vesicant bis-(beta-chloroethyl)sulfide (BCES) on the homeostasis of cell proliferation and differentiation. Lectins were used to reveal cell surface carbohydrate changes as the keratinocytes differentiate. In the newborn rat epidermis, the isolectin, Griffonia simplicifolia I-B4 (GS I-B4), binds to basal cell surfaces. Ulex europeus agglutinin I (UEA) binds to the surfaces of spinous and lower granular cells and is therefore considered an indicator of keratinocyte differentiation. A fluorometric assay was developed which determines the ratio of bound UEA to bound GS I-B4 (the UEA/B4 ratio) in primary monolayer cultures of rat cutaneous keratinocytes maintained in low Ca2+ medium. The UEA/B4 ratio was found to be a representation of the relative sizes of the differentiating and proliferating cell compartments in the monolayer cultures, respectively (W.W. Ku and I.A. Bernstein, 1988, Exp. Cell Res., 175, 298-316). Monolayer cultures exposed for 1 hr to BCES at Day 1 exhibited a dose-related increase in the UEA/B4 ratio at Day 7 when compared to solvent controls. The results from the analysis of lectin binding sites showed a decrease in GS I-B4 binding with little or no change in UEA binding as a result of BCES exposure, contributing to the increase in the UEA/B4 ratio. BCES-exposed monolayers also showed early perturbations in replicative DNA synthesis as revealed by autoradiography. Subsequent to the perturbations in replicative DNA synthesis was an inability of BCES-exposed cultures to produce cells into the monolayer through mitosis. In addition to an increase in the UEA/B4 ratio, BCES-exposed monolayers also showed a dose-related loss of DNA, with the appearance of enlarged cells at Day 7. These enlarged cells failed to show evidence of DNA synthesis, with groups of these cells showing intense UEA staining with only faint GS I-B4 staining. Overall, exposure to low concentrations of BCES appeared to disrupt the normal homeostasis of cell proliferation and differentiation in this monolayer culture system. This disruption was primarily through a reduction in the fraction of germinative (basal) cells with concomitant retention of some early differentiated cells, presumably early spinous or spinous cells.

Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes.

The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca2+ medium (0.08 mM) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56 +/- 0.05), steadily increased to a maximum of 0.84 +/- 0.09 between Days 2 and 4, and then gradually decreased to 0.41 +/- 0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [3H]thymidine into their nuclei. Electrophoresis of [3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of "96K-associated UEA-bound" radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis.

Changes in lectin binding by differentiating cutaneous keratinocytes from the newborn rat.

Surface glycoconjugates of cells from the basal layer of the skin of the newborn rat bind the isolectin I-B4 from Griffonia simplicifolia (GS I-B4) (alpha-D-galactosyl specificity). Surface glycoconjugates of the differentiated cells from the spinous and lower granular layers bind Ulex europeus agglutinin I (UEA) (alpha-L-fucosyl specificity). The change from GS I-B4 binding to UEA binding was studied in rat keratinocytes that were cultured as a monolayer in low-calcium medium until confluence, and then induced to stratify and terminally differentiate by raising the calcium concentration of the medium. The cells in the monolayer had basal cell morphology and exhibited surface binding of GS I-B4. However, at confluence, 30-40% of these cells also showed surface binding of UEA. There was an increase with time in the number of cells which bound both GS I-B4 and UEA. Raising the calcium concentration of the medium resulted in an increase in UEA binding. Cells of the upper layers of the stratifying cultures showed intense UEA binding but did not show any GS I-B4 binding. Double staining of frozen sections of newborn rat skin with fluorescein-conjugated GS I-B4 and rhodamine-conjugated UEA revealed that the surfaces of cells from the lower spinous layer bound both lectins. Thirty percent of the major glycoprotein fraction, that was isolated from the membranes of the epidermal cells of the newborn rat and was bound to an affinity column of UEA-Sepharose 4B, was also bound to an affinity column of GS I-B4-Sepharose 4B. These results indicate that surface glycoconjugates of rat keratinocytes differentiating in culture exhibit a change from GS I-B4 binding to UEA binding; the change in the cell surface glycoconjugates that results in the appearance of UEA binding, a feature of differentiated cells, occurs independently of stratification; and the change from GS I-B4 binding to UEA binding probably involves an "intermediate" glycoconjugate that binds both GS I-B4 and UEA and is found on the surface of cells from the lower spinous layer of the epidermis of the newborn rat.