Farmworker pesticide exposure and community-based participatory research: rationale and practical applications.

The consequences of agricultural pesticide exposure continue to be major environmental health problems in rural communities. Community-based participatory research (CBPR) is an important approach to redressing health disparities resulting from environmental causes. In this article we introduce a collection of articles that describe projects using CBPR to address the health disparities resulting from pesticide exposure in agricultural communities, particularly the communities of migrant and seasonal farmworkers. The articles in this collection are based on a workshop convened at the 1999 American Public Health Association meeting. The goals in presenting this collection are to provide those endeavoring to initiate CBPR projects needed information, guidelines, and procedures to improve the quality of the CBPR experience; to increase the scientific validity of CBPR projects; and to reduce the potential difficulties and stress of these collaborations. In this introduction we discuss the context in which these projects operate, summarizing background information about farmworkers in the United States, what is known about farmworker pesticide exposure, and the concept of community-based participatory research. Finally, the articles in this collection are summarized, and major themes common to successful CBPR projects are identified. These common features are taking the time to interact with the community, using multiple approaches to engage the different parts of the community, understanding different participants often have different goals, appreciating each group's strengths, valuing community knowledge, and being flexible and creative in conducting research. The final article in this collection describes the translational research program at the National Institute of Environmental Health Sciences (NIEHS) highlighting activities pertinent to the health of rural communities, giving an overview of NIEHS-supported projects addressing health concerns of Native Americans and rural African-American communities in addition to farmworkers, and discussing future plans for CBPR at NIEHS.

Commitment of the National Institute of Environmental Health Sciences to community-based participatory research for rural health.

The National Institute of Environmental Health Sciences (NIEHS) is the leading biomedical research institution in the United States whose mission is to support research that seeks to understand how environmental exposures affect human health. NIEHS possesses a longstanding interest in the health effects of agrochemical and other environmental exposures in rural America, including pesticides, to farmers and their families and to migrant farmworkers and their families. In recent years, NIEHS has begun augmenting traditional basic science investigations with innovative programs that translate findings from the laboratory to affected populations. It is through community-based participatory research that NIEHS strives to advance the public health field by fostering the development of culturally relevant interventions that will reduce exposures to environmental contaminants and the risk of environmentally induced disease. In this article, we describe the translational research program at NIEHS as it relates to the NIEHS mission and highlight activities pertinent to the health of rural communities, especially underserved populations. We provide an overview of NIEHS-supported projects addressing health concerns of Native American and rural African-American communities in addition to farmworkers. We conclude with a discussion of future plans for community-based participatory research at NIEHS.

The National Institute of Environmental Health Sciences' research program on children's environmental health.

This article highlights the wide array of research programs supported by the National Institute of Environmental Health Sciences (NIEHS) that address issues related to children's environmental health. Special attention is given to the interagency, collaborative Centers for Children's Environmental Health and Disease Prevention Research program. A brief description of each of the eight centers highlights scientific foci and research efforts to date. In addition to discussing NIEHS-supported research programs, the article emphasizes the NIEHS' commitment to the promotion of translating basic research findings into public health knowledge so that culturally sensitive and applicable interventions may be developed.

Location and molecular cloning of D1 dopamine receptor.

Recently, our laboratory has purified the D1 dopamine receptor 6600 fold to near homogeneity from digitonin solubilized rat striatal membranes using sequential affinity, ion exchange, lectin, and size exclusion chromatographies. The resulting receptor preparations still retained ligand binding activity (-11,000 pmol [3H]SCH 23390 bound per mg/protein) and appeared as a single band at 70-80 kDa on SDS-PAGE. In order to learn more about the sequence and structure of this protein, we recently cloned the gene for a human CNS D1 dopamine receptor. This gene has an open reading frame of 1388 nucleotides and encoded for a protein with a deduced amino acid sequence of 446 residues. When expressed in mammalian cells the cloned D1 receptor had all the ligand binding properties expected for a D1 receptor (SCH 23390 > cis flupenthixol > raclopride and SKF 38393 > apomorphine > dopamine > quinpirole). The cloned D1 receptor was found to stimulate adenylyl cyclase but not phospholipase C. The message for this D1 dopamine receptor was found in caudate, putamen, frontal cortex, and hippocampus, but not in substantia nigra, heart, or kidney. These accomplishments now will allow the pursuit of biochemical studies of the receptor protein as well as investigations into structure/function relationship of the receptor using a molecular biological techniques.

D2 dopamine receptors in the human retina: cloning of cDNA and localization of mRNA.

1. We have obtained a cDNA clone encoding a human retinal D2 dopamine receptor. 2. The longest open reading frame (1242 bp) of this clone encodes a protein of 414 amino acids having a predicted molecular weight of 47,000 and a transmembrane topology similar to that of other G protein-coupled receptors. 3. Transient transfection of COS-7 cells with an expression vector containing the clone resulted in expression of a protein possessing a pharmacological profile similar to that of the D2 dopamine receptor found in striatum and retina. 4. Northern blot analysis indicated that, in rat brain and retina, the mRNA for this receptor was 2.9 kb in size. 5. In situ hybridization was performed to examine the distribution of the mRNA for this receptor in human retina. Specific hybridization was detected in both the inner and the outer nuclear layers. 6. These findings are consistent with prior physiological and autoradiographic studies describing the localization of D2 dopamine receptors in vertebrate retinas. Our observations suggest that photoreceptors as well as cells in the inner nuclear layer of human retinas may express the mRNA for this D2 dopamine receptor.

Localization of D1 dopamine receptor mRNA in brain supports a role in cognitive, affective, and neuroendocrine aspects of dopaminergic neurotransmission.

Expression of a D1 dopamine receptor was examined in the rat brain by using a combination of in situ hybridization and in vitro receptor autoradiography. Cells expressing D1 receptor mRNA were localized to many, but not all, brain regions receiving dopaminergic innervation. The highest levels of hybridization were detected in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Cells expressing D1 receptor mRNA were also detected throughout the cerebral cortex, limbic system, hypothalamus, and thalamus. D1 receptor mRNA was differentially expressed in distinct regions of the hippocampal formation. Dentate granule cells were labeled in dorsal but not ventral regions, whereas the subicular complex was prominently labeled in ventral but not dorsal regions. Intermediate to high levels of D1 binding sites, but no hybridizing D1 receptor mRNA, were detected in the substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus, and subthalamic nucleus. In these brain regions, which are involved in the efferent flow of information from the basal ganglia, D1 receptors may be localized on afferent nerve terminals originating in other brain regions. These results indicate that in addition to a role in control of motor function, the D1 receptor may also participate in the cognitive, affective, and neuroendocrine effects of dopaminergic neurotransmission.

Molecular characterization of G-protein coupled receptors: isolation and cloning of a D1 dopamine receptor.

This article summarizes the recent progress our laboratory has made in understanding the molecular characteristics of the D1 dopamine receptor. The D1 dopamine receptor from rat striatum has been purified to near homogeneity using a combination of several chromatographic steps. Furthermore, the gene for the human D1 dopamine receptor has been cloned, sequenced, and expressed. The cloned receptor has all the pharmacologic and biochemical properties of the classical D1 receptor coupled to adenylyl cyclase which has been previously described in the central nervous system.

Light onset stimulates tyrosine hydroxylase activity in isolated teleost retinas.

The action of tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine, the most abundant catecholamine in vertebrate retinas. I have examined the activation and regulation of this enzyme in isolated retinas of green sunfish, Lepomis cyanellus. Exposing previously dark-adapted retinas to constant illumination for a period of 10 min increased enzymatic activity 2.2-fold over that present in retinas incubated in darkness. Thus, light onset activates tyrosine hydroxylase in teleost retinas. Stimulation of the activity of tyrosine hydroxylase under these conditions was associated with a decrease in the apparent Km of the enzyme for its pteridine cofactor without a change in the apparent Vmax of the reaction. This result suggests that short-term exposure to light increases dopamine synthesis by enhancing the affinity of the enzyme for its naturally occurring cofactor. These findings are consistent with the idea that light activates dopaminergic neurons in teleost retinas.

Molecular cloning and expression of the gene for a human D1 dopamine receptor.

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.

Molecular characterization of dopamine receptors.

The D1 and D2 dopamine receptors have been biochemically characterized using specific probes based on the subtype selective antagonists SCH 23390 and spiperone, respectively. The D2 dopamine receptor was identified from several tissues by photoaffinity labeling and was purified from bovine anterior pituitary to homogeneity using a combination of affinity, lectin and hydroxylapatite chromatography. A complementary DNA (cDNA) encoding a rat brain D2 dopamine receptor has been cloned via low stringency hybridization using a portion of the beta 2-adrenergic receptor gene as a probe. Photoaffinity crosslinking and affinity chromatography have also been used to identify and purify the rat brain D1 dopamine receptor.

Dopamine receptor subtypes: beyond the D1/D2 classification.

The D1/D2 dopamine receptor classification is widely accepted. However, intense investigative efforts over the last several years using pharmacological, biochemical and behavioral approaches have produced results that are increasingly difficult to reconcile with the existence of only two dopamine receptor subtypes. Recent developments, including cloning of the cDNAs and/or genes for several members of the large family of G-protein-coupled receptors, have revealed that heterogeneity in the pharmacological or biochemical characteristics of individual receptors often indicates the presence of previously unsuspected molecular subtypes. In this article, Marc Caron and colleagues have assembled the main lines of evidence that suggest the presence of several novel subtypes for both D1 and D2 dopamine receptors and predict that molecular cloning will, in the near future, confirm their existence.

Dopamine induces light-adaptive retinomotor movements in bullfrog cones via D2 receptors and in retinal pigment epithelium via D1 receptors.

In the eyes of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) exhibit characteristic retinomotor movements in response to changes in ambient illumination and to signals from an endogenous circadian clock. We previously reported that 3,4-dihydroxyphenylethylamine (dopamine) mimicked the effect of light on these movements in photo-receptors and RPE cells of green sunfish, Lepomis cyanellus, by interacting with D2 dopaminergic receptors. Here, we report that dopamine also mimics the effect of light on cone and RPE retinomotor movements in bullfrogs, Rana catesbeiana, i.e., dopamine induces cone contraction and RPE pigment dispersion. Dopamine induced cone contraction in isolated dark-adapted bullfrog retinas incubated in constant darkness in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This effect of dopamine was inhibited by a D2 but not a D1 antagonist and mimicked by a D2 but not a D1 agonist. These results suggest that induction of cone contraction by dopamine is mediated by D2 dopaminergic receptors and that cone adenylate cyclase activity is inhibited. Thus, dopamine acts via the same type of receptor in both bullfrog and green sunfish retinas to induce cone contraction. In contrast, dopamine influences RPE retinomotor movement via different receptors in fish and bullfrog. Dopamine induced light-adaptive pigment dispersion in isolated dark-adapted bullfrog RPE-eyecups incubated in constant darkness in normal Ringer's solution. Because the retina was not present, these experiments demonstrate a direct effect of dopamine on bullfrog RPE. This effect of dopamine on bullfrog RPE was inhibited by a D1 but not a D2 antagonist and mimicked by a D1 but not a D2 agonist. Furthermore, agents that increase the concentration of intracellular cyclic AMP also induced pigment dispersion in dark-adapted bullfrog RPE-eyecups incubated in the dark. These results suggest that dopamine induces pigment dispersion in bullfrog RPE via D1 dopaminergic receptors. Thus, dopamine acts via different receptors on bullfrog (D1) versus green sunfish (D2) RPE to induce pigment dispersion. In addition, inhibitor studies indicate that pigment dispersion is actin dependent in teleost but not in bullfrog RPE. Dopamine-induced pigment dispersion was inhibited by cytochalasin D in isolated RPE sheets of green sunfish but not in RPE-eyecups of bullfrogs. Together, these observations indicate that dopamine mimics the effect of light on cone and RPE retinomotor movements in both fish and bullfrogs. However, in the RPE, different receptors mediate the effect of dopamine, and different cytoskeletal mechanisms are used to affect pigment transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Light-induced dopamine release from teleost retinas acts as a light-adaptive signal to the retinal pigment epithelium.

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules migrate in and out of the cells' long apical projections in response to changes in light condition. When the RPE is in its normal association with the retina, light onset induces pigment granules to disperse into the apical projections; dark onset induces pigment granules to aggregate into the cell bodies. However, when the RPE is separated from the retina, pigment granule movement in the isolated RPE is insensitive to light onset. It thus seems likely that a signal from the retina communicates light onset to the RPE to initiate pigment dispersion. We have examined the nature of this retina-to-RPE signal in green sunfish, Lepomis cyanellus. In isolated retinas with adherent RPE, light-induced pigment dispersion in the RPE is blocked by treatments known to block Ca2+-dependent transmitter release in the retina. In addition, the medium obtained from incubating previously dark-adapted retinas in the light induces light-adaptive pigment dispersion when added to isolated RPE. In contrast, the medium obtained from incubating dark-adapted retinas in constant darkness does not affect pigment distribution when added to isolated RPE. These results are consistent with the idea that RPE pigment dispersion is triggered by a substance that diffuses from the retina at light onset. The capacity of the conditioned medium from light-incubated retinas to induce pigment dispersion in isolated RPE is inhibited by a D2 dopamine antagonist, but not by D1 or alpha-adrenergic antagonists. Light-induced pigment dispersion in whole RPE-retinas is also blocked by a D2 dopamine antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of distinct D2 dopaminergic and alpha 2-adrenergic receptors induces light-adaptive pigment dispersion in teleost retinal pigment epithelium.

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4-dihydoxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half-maximal effect at approximately 2 nM; clonidine, at 1 microM. Dopamine-induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by D1 or alpha-adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a D1 dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine-induced dispersion was inhibited by the alpha 2-adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and alpha 2-adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark-adaptive pigment aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian rhythms in the green sunfish retina.

We investigated the occurrence of circadian rhythms in retinomotor movements and retinal sensitivity in the green sunfish, Lepomis cyanellus. When green sunfish were kept in constant darkness, cone photoreceptors exhibited circadian retinomotor movements; rod photoreceptors and retinal pigment epithelium (RPE) pigment granules did not. Cones elongated during subjective night and contracted during subjective day. These results corroborate those of Burnside and Ackland (1984. Investigative Ophthalmology and Visual Science. 25:539-545). Electroretinograms (ERGs) recorded in constant darkness in response to dim flashes (lambda = 640 nm) exhibited a greater amplitude during subjective night than during subjective day. The nighttime increase in the ERG amplitude corresponded to a 3-10-fold increase in retinal sensitivity. The rhythmic changes in the ERG amplitude continued in constant darkness with a period of approximately 24 h, which indicates that the rhythm is generated by a circadian oscillator. The spectral sensitivity of the ERG recorded in constant darkness suggests that cones contribute to retinal responses during both day and night. Thus, the elongation of cone myoids during the night does not abolish the response of the cones. To examine the role of retinal efferents in generating retinal circadian rhythms, we cut the optic nerve. This procedure did not abolish the rhythms of retinomotor movement or of the ERG amplitude, but it did reduce the magnitude of the nighttime phases of both rhythms. Our results suggest that more than one endogenous oscillator regulates the retinal circadian rhythms in green sunfish. Circadian signals controlling the rhythms may be either generated within the eye or transferred to the eye via a humoral pathway.

Dopaminergic regulation of cone retinomotor movement in isolated teleost retinas: I. Induction of cone contraction is mediated by D2 receptors.

In the retinas of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo characteristic movements in response to changes in light intensity and to signals from an endogenous circadian clock. To identify agents responsible for mediating light and/or circadian regulation of these retinomotor movements, we investigated the effects of hormones and neurotransmitters on cone, rod, and RPE movements in the green sunfish, Lepomis cyanellus. We report here that 3,4-dihydroxyphenylethylamine (dopamine) mimics the effect of light by inducing light-adaptive retinomotor movements in all three cell types. In isolated dark-cultured retinas, dopamine induced light-adaptive cone contraction with a half-maximal effect at 10(-8) M. This effect of dopamine was inhibited by antagonists with a potency order characteristic of D2 receptor mediation. The dopamine uptake blocker benztropine also induced light-adaptive cone contraction in isolated dark-cultured retinas, suggesting that there is continuous dopamine release in the dark but that concomitant uptake normally prevents activation of cone contraction. That dopamine plays a role in light regulation of cone movement is further suggested by the observation that light-induced cone contraction was partially inhibited by sulpiride, a selective D2 dopamine antagonist, or by Co2+, a blocker of synaptic transmission. Sulpiride also promoted dark-adaptive cone elongation in isolated light-adapted retinas, suggesting that continuous dopamine action is required in the light to maintain the light-adapted cone position. Dopamine can act directly on D2 receptors located on rod and cone inner/outer segments: dopamine induced light-adaptive retinomotor movements in isolated distal fragments of dark-adapted photoreceptors cultured in the dark. Together our results indicate that dopamine induces light-adaptive retinomotor movements in cones, rods, and RPE cells by activating D2 receptors. We suggest that, in vivo, dopamine plays a role in both light and circadian regulation of retinomotor movements.

Dopaminergic regulation of cone retinomotor movement in isolated teleost retinas: II. Modulation by gamma-aminobutyric acid and serotonin.

In the accompanying paper we reported that 3,4-dihydroxyphenylethylamine (dopamine) induced light-adaptive retinomotor movements in teleost photoreceptors and that this effect was mediated by D2 dopamine receptors located on the photoreceptors themselves. In this study, we investigated the effects on cone retinomotor movement of three agents that have been reported by others to modulate retinal dopamine release: gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT, serotonin), and melatonin. We report here that the GABA antagonists bicuculline and picrotoxin induced light-adaptive cone contraction in dark-adapted green sunfish retinas cultured in constant darkness; thus they mimic the effect of light or exogenously applied dopamine. Since their effects were blocked by either the D2 dopamine antagonist sulpiride or by Co2+, it seems likely that these agents act by enhancing retinal dopamine release. The GABA agonist muscimol produced effects opposite to those of GABA antagonists. Muscimol inhibited light-induced cone contraction in previously dark-adapted retinas and induced dark-adaptive cone elongation in light-adapted retinas. These results suggest that in green sunfish retinas, as has been reported for other retinas, GABA inhibits dopamine release. 5-HT induced light-adaptive cone contraction in dark-adapted retinas; thus 5-HT also mimics the effect of light or exogenously applied dopamine. The effect of 5-HT was blocked by sulpiride, Co2+, or the 5-HT antagonist mianserin. These results suggest that 5-HT induces cone contraction by stimulating dopamine release. Melatonin neither inhibited dopamine-induced cone contraction in retinas cultured in the dark nor induced cone elongation in retinas cultured in the light. Our results suggest that both GABA and 5-HT (but not melatonin) affect cone retinomotor movements in green sunfish by modulating dopamine release: GABA by inhibiting and 5-HT by stimulating dopamine release. We report in the companion paper that dopamine induced contraction in isolated cone fragments. Together these observations strongly suggest that dopamine serves as the final extracellular messenger directly inducing light-adaptive cone retinomotor movement, and that GABA and 5-HT affect these movements by modulating dopamine release.

Dopamine inhibits forskolin- and 3-isobutyl-1-methylxanthine-induced dark-adaptive retinomotor movements in isolated teleost retinas.

We have been investigating the mechanisms of diurnal and circadian regulation of teleost retinomotor movements. In the retinas of lower vertebrates, photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo movements at dawn and dusk. These movements continue to occur at subjective dawn and dusk in animals maintained in constant darkness. Cone myoids contract at dawn and elongate at dusk; RPE pigment disperses into the epithelial cells' long apical processes at dawn and aggregates into the cell bodies at dusk. We report here that forskolin, an adenylate cyclase activator, and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, each induces dark-adaptive cone and RPE retinomotor movements in isolated light-adapted green sunfish retinas cultured in constant light. Forskolin induces a 22-fold elevation in retinal cyclic AMP content. Forskolin- and IBMX-induced movements are inhibited approximately 65% and 95%, respectively, by 3,4-dihydroxyphenylethylamine (dopamine). However, dopamine does not inhibit dark-adaptive movements induced by dibutyryl cyclic AMP. Epinephrine is much less effective than dopamine in inhibiting forskolin-induced movements, while phenylephrine and clonidine are totally ineffective. These results are consistent with our previous findings that treatments that increase intracellular cyclic AMP content promote dark-adaptive retinomotor movement. They further suggest that dopamine inhibits adenylate cyclase activity in photoreceptors and RPE cells and thereby favors light-adaptive retinomotor movements.