Evaluation of temperature- and ethanol-related developmental degree variations by a new scoring system (FETAX-score) applicable to Frog Embryo Teratogenicity Assay: Xenopus.

The aim of the present work is to propose a new quantitative assessment method (FETAX-score) for determining the degree of Xenopus laevis embryo development intended for use in embryotoxicity studies. Inspired by a similar scoring system used to evaluate developmental delays (young-for-age phenotypes) in rat embryos cultured in vitro, the FETAX-score was established by considering seven morphological features (head, naris, mouth, lower jaw, tentacles, intestine, anus) that are easily evaluable in tadpoles during the late stages of development at the conclusion of the test. Given that X. laevis development is temperature-dependent and that temperatures below 14°C and above 26°C are teratogenic, the FETAX-score was tested in embryos maintained at 17, 20, 23 and 26°C. No abnormalities were observed in any group, while the total score was temperature-related, suggesting that the FETAX-score is sensitive to moderate distress that does not influence general morphology. Intestine and anus were the least sensitive structures to temperature variations. To assess the applicability of the FETAX-score in developmental toxicological studies, we evaluated FETAX-score in tadpoles exposed during the morphogenetic period to Ethanol (Eth) at concentrations of 0, 0.25, 0.5, 1, 1.5, and 2 % v/v. Gross malformations were observed only in tadpoles from the Eth 2 % group. By contrast, data analysis of the other Eth groups showed dose-related reductions in the FETAX-score. Tentacles were the most sensitive structures to Eth-related delays. These results support the use of the FETAX-score to quantitatively assess developmental deviations in FETAX embryotoxicity studies.

Effects of combined exposure to two bisphenol plasticizers (BPA and BPB) on Xenopus laevis development.

Due to its endocrine disruptive activity, the plastic additive Bisphenol A (BPA) is classified as substance of very high concern (EU ECHA 2017). A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species including the amphibian Xenopus laevis, where severe branchial defects were associated to lethality. The exposure of X. laevis embryos to the BPA analogue bisphenol B (BPB) was recently linked to similar teratogenic effects, with BPB having relative potency about 3 times higher than BPA. The combined BPA-BPB exposure is realistic as both BPA and BPB are detected in human samples and environment. Limited experimental data are available on the combined developmental toxicity of BPA and BPB. The aim of the present work is to evaluate the effects of BPA and BPB mixture in the X. laevis development model, using R-FETAX procedure. The exposure was limited to the first day of development (corresponding to the phylotypic developmental period, common to all vertebrates). Samples were monitored for lethal effects during the full six-day test period and the external morphology was evaluated at the end of the test. Mixture effects were described by modelling, using the PROAST software package. Overall data modelling showed that dose-addiction could not be rejected, suggesting a health concern for co-exposure.

Teratogenic and neuro-behavioural toxic effects of bisphenol A (BPA) and B (BPB) on Xenopus laevis development.

Bisphenol A (BPA) is a plastic additive with endocrine disruptive activity, classified in 2017 by EU ECHA as substance of very high concern. A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species, including the amphibian Xenopus laevis. Among BPA analogues, bisphenol B (BPB) is used as alternative in different not-EU countries, including US, but seems to share with BPA its endocrine disruptor properties. Aim of the present work is the evaluation of the effects of BPB versus BPA exposure in a X. laevis developmental model. A windowed exposure (R-FETAX method) was applied covering the developmental phylotypic period (teratogenicity window), or the late tailbud stages (neuro-behavioural toxicity window, corresponding to the spontaneous swimming acquisition period). Samples were monitored for lethal effects during the full test period. External morphology evaluation and deglutition functional test were applied in any group. Abnormal tadpoles were also processed for cartilage staining. In groups exposed during neuro-behavioural toxicity window the swimming test was also applied. Lethality and malformations were obtained only in samples exposed during the teratogenicity window; these data were modelled using PROAST software and BPB relative potency resulted about 3 times higher than BPA. The day-by-day evaluation revealed that lethality was correlated to embryonic abnormal development of gills and apoptosis in gill primordia. Teratogenicity was never detected in groups exposed during the neuro-behavioural toxicity window, where some significant neuro-behavioural deficits were detected in tadpoles exposed to the highest tested concentrations of BPA and BPB.

CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells.

The prototypical genetic autoimmune disease is immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a severe pediatric disease with limited treatment options. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which plays a critical role in immune regulation. As a monogenic disease, IPEX is an ideal candidate for a therapeutic approach in which autologous hematopoietic stem and progenitor (HSPC) cells or T cells are gene edited ex vivo and reinfused. Here, we describe a CRISPR-based gene correction permitting regulated expression of FOXP3 protein. We demonstrate that gene editing preserves HSPC differentiation potential, and that edited regulatory and effector T cells maintain their in vitro phenotype and function. Additionally, we show that this strategy is suitable for IPEX patient cells with diverse mutations. These results demonstrate the feasibility of gene correction, which will be instrumental for the development of therapeutic approaches for other genetic autoimmune diseases.

A glyphosate micro-emulsion formulation displays teratogenicity in Xenopus laevis.

Glyphosate is the active ingredient in broad-spectrum herbicide formulations used in agriculture, domestic area and aquatic weed control worldwide. Its market is growing steadily concurrently with the cultivation of glyphosate-tolerant transgenic crops and emergence of weeds less sensitive to glyphosate. Ephemeral and lentic waters near to agricultural lands, representing favorite habitats for amphibian reproduction and early life-stage development, may thus be contaminated by glyphosate based herbicides (GBHs) residues. Previous studies on larval anuran species highlighted increased mortality and growth effects after exposure to different GBHs in comparison to glyphosate itself, mainly because of the surfactants such as polyethoxylated tallow amine present in the formulations. Nevertheless, these conclusions are not completely fulfilled when the early development, characterized by primary organogenesis events, is considered. In this study, we compare the embryotoxicity of Roundup® Power 2.0, a new GBH formulation currently authorized in Italy, with that of technical grade glyphosate using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Our results evidenced that glyphosate was not embryolethal and only at the highest concentration (50 mg a.e./L) caused edemas. Conversely, Roundup® Power 2.0 exhibited a 96 h LC50 of 24.78 mg a.e./L and a 96 h EC50 of 7.8 mg a.e./L. A Teratogenic Index of 3.4 was derived, pointing out the high teratogenic potential of the Roundup® Power 2.0. Specific concentration-dependent abnormal phenotypes, such as craniofacial alterations, microphthalmia, narrow eyes and forebrain regionalization defects were evidenced by gross malformation screening and histopathological analysis. These phenotypes are coherent with those evidenced in Xenopus laevis embryos injected with glyphosate, allowing us to hypothesize that the teratogenicity observed for Roundup® Power 2.0 may be related to the improved efficacy in delivering glyphosate to cells, guaranteed by the specific surfactant formulation. In conclusion, the differences in GBH formulations should be carefully considered by the authorities, since sub-lethal and/or long-term effects (e.g. teratogenicity) can be significantly modulated by the active ingredient salt type and concentration of the adjuvants. Finally, the mechanistic toxicity of glyphosate and GBHs are worthy of further research.

Ectopic FOXP3 Expression Preserves Primitive Features Of Human Hematopoietic Stem Cells While Impairing Functional T Cell Differentiation.

FOXP3 is the transcription factor ruling regulatory T cell function and maintenance of peripheral immune tolerance, and mutations in its coding gene causes IPEX autoimmune syndrome. FOXP3 is also a cell-cycle inhibitor and onco-suppressor in different cell types. In this work, we investigate the effect of ectopic FOXP3 expression on HSC differentiation and we challenged this approach as a possible HSC-based gene therapy for IPEX. FOXP3-expressing HSC showed reduced proliferation ability and increased maintenance of primitive markers in vitro in both liquid and OP9-ΔL1 co-cultures. When transplanted into immunodeficient mice, FOXP3-expressing HSC showed significantly enhanced engraftment ability. This was due to a pronounced increase in the frequency of repopulating cells, as assessed by extreme limiting dilution assay. Likely underlying the increased repopulating ability, FOXP3 expressing HSC showed significantly enhanced expression of genes controlling stemness features. However, peripheral T cells developed in the FOXP3-humanized mice were quantitatively reduced and hyporesponsive to cytokine and polyclonal stimulation. Our findings reveal unpredicted effects of FOXP3 in the biology of HSC and may provide new tools to manipulate primitive features in HSC for clinical applications. Moreover, they formally prove the need of preserving endogenous FOXP3 regulation for an HSC-based gene therapy approach for IPEX syndrome.

Anti-infective prophylaxis for primary immunodeficiencies: what is done in Italian Primary Immunodeficiency Network centers (IPINet) and review of the literature.

Primary immunodeficiencies (PIDs) are rare diseases characterized by an increased susceptibility to infections. Early diagnosis and appropriate treatment are critical for reducing morbidity and mortality. Based on available data, the efficacy of antibiotic administration for the prophylaxis of infections remains uncertain, and recommendations supporting this practice are poor. The use of antimicrobial prophylaxis is mainly based on single institution-specific experience without controlled measurements of patient safety and quality health outcomes. To address this issue an Italian Network on Primary Immunodeficiencies (IPINet) has been set up in 1999 within the Italian Association of Pediatric Hematology and Oncology (AIEOP) to increase the awareness of these disorders among physicians. Further, diagnostic and treatment guideline recommendations have been established to standardize the best clinical assistance to all patients, including antibiotic prophylaxis, and for a national epidemiologic monitoring of PIDs. The aim of this review is not only to give a scientific update on the use of antimicrobial prophylaxis in selected congenital immunological disorders but also to draw a picture of this practice in the context of the Italian Primary Immunodeficiency Network (IPINet). Controlled multicenter studies are necessary to establish if, when and how you should start an efficacious antimicrobial prophylaxis.

Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome.

Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.

Mitochondrial DNA sequence variations in some Italian wild boar populations.

In order to investigate the relationships between Italian wild boar and major pig breeds, we studied the genetic variability of four wild boar populations in Italy (Arezzo, Pisa, Parma, Bergamo) using a 533-bp fragment of the mitochondrial control region. Sixty-nine wild boar samples were analysed, allowing the identification of 10 distinct haplotypes, which involve a total of 15 single nucleotide polymorphisms. Phylogenetic and network analyses were performed also considering several sequences of wild and domesticated forms available in the databases. The Bayesian phylogenetic tree and the Median-Joining network analyses show three main groups: the Italian (IT), European (EU) and Asian (AS) clades. The IT clade corresponds to the Maremma endemic wild boar population and also includes Sardinian individuals, while the EU and AS groups include wild boars as well as domestic pig breeds. Only two individuals from Pisa cluster in the IT group, whereas two haplotypes from Bergamo cluster in the AS group and all other samples cluster in the EU clade. These findings suggest that in Italy wild boar populations have a mixed origin, both EU and AS, and that an interbreeding between wild and domesticated strains has probably occurred. Eight of the 10 wild boars coming from the Migliarino-San Rossore-Massaciuccoli Regional Park (Pisa) belong to H2 and H3 haplotypes, and cluster into the EU clade, suggesting that this regional park is not anymore exclusive of the endemic Maremma wild boar.

Histological changes and micronucleus induction in the zebra mussel Dreissena polymorpha after paraquat exposure.

The herbicide paraquat (PQ), still widely used in developing countries, represents a serious risk factor for human and environmental health. To test the sublethal effects of PQ on the freshwater bivalve Dreissena polymorpha, mussels were exposed to 0.125, 0.250, 0.500 mg/L for 7 and 14 days and histologically screened. PQ's genotoxic potential was also determined in haemocytes by the micronucleus, MN, assay. At concentrations > or = 0.250 mg/L, severe lesions, such as cellular vacuolation, lysis and thinness of the germinative epithelia were observed in the digestive gland and testis. A positive trend between the number of granulocytes and all PQ concentrations was observed in both gonads and digestive glands, addressing the inflammatory capacity of this herbicide on these tissues. Mussels exposed to PQ also exhibited a significant MN induction. The spontaneous MN frequencies ranged from 2.75 to 0.425 per cent, while PQ-induced MN rates in treated mussels were between 3.50 and 1.250 per cent. The histopathological effects on the digestive and reproductive systems, as well as the MN induction in the haemocytes, confirmed the cytotoxic and genotoxic effects of PQ also in D. polymorpha.

Histopathological effects induced by paraquat during Xenopus laevis primary myogenesis.

The oxidative agent paraquat induced tail abnormalities during Xenopus laevis development. Specimens exposed from blastula to the tadpole stage revealed pear-shaped myocytes and irregular intersomitic boundaries. The histological feature of the axial musculature was evaluated in embryos sampled at significant stages of the primary myogenesis. During the somitogenesis PQ-treated embryos showed normal appearing myotomes, but reduced PAS activity in the post-rotating myotomal cells, and myoblasts with slight vacuolations. Once etched from the vitelline envelope, embryos showed severely altered myoblasts with irregular cellular apexes, heavy sarcoplasmic vacuolations, pyknotic nuclei and disorganizing intersomitic boundaries. Myotomes with many necrotic myocytes containing disorganized contractile material and heavily malformed intersomitic boundaries characterized the late myogenic stages. Our results evidence the heaviest PQ histopathological effects to affect myogenesis of post-etched embryos, suggesting a possible linkage between the swimming activity and the oxidative damage to muscle tissue.

Utilizing regulatory T cells to control alloreactivity.

Effective resetting of the immune system cannot be achieved by non-specific immunosuppression. Instead, novel strategies aim at harnessing the body's natural tolerance mechanisms to rectify an Ag-specific response without disturbing other immune functions. Fine-tuning of the balance between Ag-specific effector and regulatory T (Tr) cells is a promising strategy that requires detailed understanding of the differentiation and expansion pathways of the relevant Tr cell subsets. Here we review recent developments regarding the control of alloreactivity by induction and expansion of Tr cells. T-cell activation in the presence of tolerogenic APC and cytokines leads to the induction of Tr cells, which can mediate tolerance through cytokine-dependent and/or contact-dependent mechanisms. Better understanding of the mechanisms of immune regulation mediated by Tr cells may enable fine-tuning of specific immune responses and pave the way for novel therapeutic approaches.

Induction of transplantation tolerance in humans using fetal cell transplants.

When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.

OOCYTE DEGENERATION AND ALTERED OVIPOSITORY ACTIVITY INDUCED BY PARAQUAT IN THE FRESHWATER SNAIL PHYSA FONTINALIS (GASTROPODA: PULMONATA).

The freshwater snail Physa fontinalis was used as a bioindicator to study the effects of the herbicide Paraquat (PQ) in laboratory assays. The test solutions used, 0.125, 0.25, and 0.5 mg/l PQ, were in the range of the concentrations recommended for aquatic weed control. The study was carried out in two stages to determine the influence of PQ on the ovipository activity of Physa fontinalis, and the histological effects on these snails. Specimens exposed to PQ continued to be reproductively active, but the number of egg masses and eggs laid decreased significantly. Mortality was almost the same in all the experimental lots, but was significantly related to the production of egg masses only in the controls. The histological analysis showed a clear trend among PQ concentrations and degenerating oocytes, but no visible effects on the male sex-line were observed. By interfering with fertility, PQ has an action that may go well beyond its lethal effect on individuals, suggesting that this herbicide should be strictly regulated in weed control programmes. Moreover, since PQ was observed to interfere with the reproductive process, its endocrine disrupting action must not be excluded.

Type 1 T regulatory cells.

Suppression by T regulatory (Tr) cells is essential for induction of tolerance. Many types of Tr cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which these cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell-to-cell interactions and/or the production of interleukin (IL)-10 and transforming growth factor (TGF)-beta. Type-1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-beta. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL-15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL-10 and TGF-beta. Further characterisation of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen.

DDT contamination in Lake Maggiore (N. Italy) and effects on zebra mussel spawning.

Zebra mussel (Dreissena polymorpha) is commonly used as contaminant bioindicator in Europe and North America. We used the zebra mussel to follow DDT pollution trends from 1996 to 1997 in Pallanza bay, Lake Maggiore, near the inlet of the River Toce, after a DDT-manufacturing plant discharging residues into a tributary of the River Toce had closed down. DDT contamination fell off sharply outside the bay, but tissue concentrations of the parent compound and residues remained high in molluscs sampled within the bay a year later. Molluscs collected in Pallanza bay in June 1997 released gametes earlier than those sampled at a nearby reference station. Histological studies showed that a significant percentage of these specimens showed marked oocyte degeneration suggesting that DDTs have endocrine-disrupting effects in this species.

Paraquat embryotoxicity in the Xenopus laevis cleavage phase.

The high Paraquat (PQ, 1-1'-dimethyl-4,4'bipyridylium dichloride) embryotoxicity in Xenopus laevis has been shown to be due to its rapid reduction and instantaneous re-oxidation which produces a reactive oxygen species, ROS. Nevertheless, PQ did not show any effects before hatching, stage 32, which showed a resistance, in early X. laevis development, to oxidative damage. Moreover, in view of its genotoxic properties in several experimental models, we studied PQ in the X. laevis cleavage phase that, characterized by a series of rapid mitotic divisions, might be damaged by genotoxic compounds. Embryos were exposed to 20, 40, 60, and 80 mg/l PQ concentrations from stage 2 to stage 9, and then left to develop in control FETAX solution until stage 47. The 80 mg/l PQ concentration gave 19% embryo mortality at the end of the exposure time, and 16.7% larvae mortality at the end of the test; both values were statistically different from the control, 5 and 6.8% respectively. These results confirmed the high resistance in early X. laevis development to PQ oxidative damage. The malformed larva percentages in the PQ exposed groups were higher as regards the control value but did not show any concentration-response; the most frequent malformed larvae found were affected by abnormal tail flexure coupled with abnormal gut coiling. A further experiment was carried out using the same methodology, but exposing embryos only to the 80 mg/l PQ concentration. The surviving blastulae were embedded in Paraplast, then the slides were stained with 4',6-diamidino-2-phenylindole (DAPI) and the nuclei were examined with a confocal microscope. This new preliminary procedure did not reveal any significant presence of micronucleated micromeres in PQ exposed blastulae with respect to the control. Nevertheless, the mechanism by which PQ induced abnormal tail flexure after cleavage exposure remained unknown. PQ seemed to pass through the jelly coats and vitelline membrane, but it expressed teratogenicity between the 2nd and 3rd day. PQ might be accumulated in the embryos during the exposure, and might express teratogenicity later, but it did not seem to induce genotoxicity during the cleavage phase of X. laevis even at very high concentrations.

Reduction in paraquat embryotoxicity by ascorbic acid in Xenopus laevis.

The toxicity of herbicide Paraquat (PQ, 1-1'-dimethyl-4,4'bipyridylium dichloride) in animal cells is related to its rapid reduction and instantaneous reoxidation to produce the reactive oxygen species. Recently, the PQ evaluation with the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) showed its high embryotoxicity. Supposing that the embryos' death was due to PQ-related oxidative damage, we used ascorbic acid (AA), a well known antioxidant, to reduce the PQ embryotoxicity in Xenopus laevis. Embryos were exposed from stage 8 to 47 to 0.1 mg/l PQ alone, and to PQ with AA concentrations ranging from 20 to 200 mg/l, using the FETAX procedure. PQ caused 72.2% mortality, while 17.1% of surviving larvae were affected by abnormal tail flexure. The PQ mortality percentages were reduced in a clear concentration-response by up to 15.2% in the group exposed to PQ with 200 mg/l AA. The histopathologic diagnoses revealed abnormal notochord flexure coupled with vesiculated, pear-shaped myocytes only in the PQ group. After embryo exposure to PQ with 200 mg/l AA, restoration of normal axial tail structures was evident. In conclusion, PQ embryotoxicity in X. laevis was most likely due to oxidative damage that was drastically reduced by AA.

Paraquat induced embryotoxicity on Xenopus laevis development.

Paraquat (PQ, 1-1'-dimethyl-4,4'-bipyridylium dichloride) is an effective herbicide widely used in agriculture with a rate of application for aquatic weed control ranging from 0.1 to 2 parts per million. Considering its wide-spread presence in Italian wetlands, we studied its embryotoxic effects with the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The percentage of mortality as well as the percentage of malformed larvae was investigated by probit analysis. The results showed that PQ was highly embryolethal. From a LC(50) of 0.138 mg/l and TC(50) of 0.267 mg/l, a TI(50) of 0.52 was derived; indicating that PQ is to be considered a non-teratogenic compound. Remarkable was the presence of a specific malformation, classified as ventral tail flexure, in the 29% of living larvae exposed to 0.125 mg/l PQ concentration. Their histological examination showed several zones of abnormal somites containing severely affected myocytes. This confirmed the molecular mechanism of PQ toxicity in cell microfilaments. Even at the lowest concentration of 0.0625 mg/l, the difference between the mean head-tail length of control and exposed larvae was statistically significant, a sign of growth retardation. All our data emphasize that PQ must be consider highly embryotoxic on amphibian development, and suggest that this herbicide should be strictly regulated in weed control programs.