Dose-dependent Growth Retardation Research Articles

The IGF-1R Inhibitor NVP-AEW541 Causes Insulin-Independent and Reversible Cardiac Contractile Dysfunction.

The antitumor treatment NVP-AEW541 blocks IGF-1R. IGF-1R signaling is crucial for cardiac function, but the cardiac effects of NVP-AEW541 are ill defined. We assessed NVP-AEW541′s effects on cardiac function and insulin response in vivo and in isolated working hearts. We performed a dose–response analysis of NVP-AEW541 in male, 3-week-old rats and assessed the chronic effects of the clinically relevant dose in adult rats. We performed glucose tolerance tests and echocardiography; assessed the expression and phosphorylation of InsR/IGF-1R and Akt in vivo; and measured substrate oxidation, contractile function, and insulin response in the isolated working hearts. NVP-AEW541 caused dose-dependent growth retardation and impaired glucose tolerance in the juvenile rats. In the adults, NVP-AEW541 caused a continuously worsening depression of cardiac contractility, which recovered within 2 weeks after cessation. Cardiac Akt protein and phosphorylation were unchanged and associated with InsR upregulation. An acute application of NVP-AEW541 in the working hearts did not affect cardiac power but eliminated insulin’s effects on glucose and fatty acid oxidation. The systemic administration of NVP-AEW541 caused dose- and time-dependent impairment of glucose tolerance, growth, and cardiac function. Because cardiac insulin signaling was maintained in vivo but absent in vitro and because contractile function was not affected in vitro, a direct link between insulin resistance and contractile dysfunction appears unlikely.

New Insights Into the Activity of Apple Dihydrochalcone Phloretin: Disturbance of Auxin Homeostasis as Physiological Basis of Phloretin Phytotoxic Action.

Apple species are the unique naturally rich source of dihydrochalcones, phenolic compounds with an elusive role in planta, but suggested auto-allelochemical features related to “apple replant disease” (ARD). Our aim was to elucidate the physiological basis of the phytotoxic action of dihydrochalcone phloretin in the model plant Arabidopsis and to promote phloretin as a new prospective eco-friendly phytotoxic compound. Phloretin treatment induced a significant dose-dependent growth retardation and severe morphological abnormalities and agravitropic behavior in Arabidopsis seedlings. Histological examination revealed a reduced starch content in the columella cells and a serious disturbance in root architecture, which resulted in the reduction in length of meristematic and elongation zones. Significantly disturbed auxin metabolome profile in roots with a particularly increased content of IAA accumulated in the lateral parts of the root apex, accompanied by changes in the expression of auxin biosynthetic and transport genes, especially PIN1, PIN3, PIN7, and ABCB1, indicates the role of auxin in physiological basis of phloretin-induced growth retardation. The results reveal a disturbance of auxin homeostasis as the main mechanism of phytotoxic action of phloretin. This mechanism makes phloretin a prospective candidate for an eco-friendly bioherbicide and paves the way for further research of phloretin role in ARD.

Mechanism of nanotoxicity in Chlorella vulgaris exposed to zinc and iron oxide.

Usage of nanoparticle in various products has increased tremendously in the recent past. Toxicity of these nanoparticles can have a huge impact on aquatic ecosystem. Algae are the ideal organism of the aquatic ecosystem to understand the toxicity impact of nanoparticles. The present study focuses on the toxicity evaluation of zinc oxide (ZnO) and iron oxide (Fe2O3) nanoparticles towards freshwater microalgae, Chlorella vulgaris. The dose dependent growth retardation in Chlorella vulgaris is observed under ZnO and Fe2O3 nanoparticles and nanoform attributed more toxicity than their bulk counterparts. The IC50 values of ZnO and Fe2O3 nanoparticles was reported at 0.258 mg L-1 and 12.99 mg L-1 whereas, for the bulk-form, it was 1.255 mgL-1 and 17.88 mg L-1, respectively. The significant decline in chlorophyll content and increase in proline content, activity of superoxide dismutase and catalase, indicated the stressful physiological state of microalgae. An increased lactate dehydrogenase level in treated samples suggested membrane disintegration by ZnO and Fe2O3 nanoparticles. Compound microscopy, scanning electron microscopy and transmission electron microscopy confirm cell entrapment, deposition of nanoparticles on the cell surface and disintegration of algal cell wall. Higher toxicity of nanoform in comparison to bulk chemistry is a point of concern.

Isoliquiritigenin as an antioxidant phytochemical ameliorates the developmental anomalies of zebrafish induced by 2,2′,4,4′-tetrabromodiphenyl ether

2,2′,4,4′-Tetrabromodiphenyl ether (BDE47) is the most abundant PBDE congeners in biological samples. It has strong tendencies to bioaccumulate and potentially endangers development of mammals through oxidative stress. Isoliquiritigenin (ISL), an emerging natural chalcone-type flavonoid, possesses various biological and pharmacological properties, including antioxidant, anti-allergic, anti-inflammatory, anti-tumor and estrogenic activities. The purpose of the study is to explore the antioxidant effect of ISL on the amelioration of developmental anomalies induced by BDE47. Zebrafish (Danio rerio) embryos were exposed to BDE47 (1 and 10 μM) and/or ISL (4 μM) for 4 to 120 hours post fertilization (hpf), and the morphology, development, behavior, oxidative stress status and related genes expression were assessed. The results showed that BDE47 contributed to dose-dependent growth retardation and deformities, including delayed hatching, spinal curvature, reduced body length, increased death rate, aberrant behaviors and impaired dark-adapted vision, which were significantly mitigated by ISL. Besides, ISL ameliorated excessive ROS accumulation, and exaggerated the expressions of apoptosis-related genes p53, Bcl-2, caspase 3 and caspase 9 induced by BDE47, suggesting that ISL protected zebrafish from the developmental toxicity of BDE47 by inactivation of programmed apoptosis and activation of antioxidant signaling pathways. Taken together, developing ISL as a dietary supplement might be a promising preventive strategy for the amelioration of developmental toxicity induced by environmental pollutants.

Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway.

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

Cytotoxicity of carteolol to human corneal epithelial cells by inducing apoptosis via triggering the Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway

Carteolol is a frequently used nonselective β-adrenoceptor antagonist for glaucoma and ocular hypertension treatment, and its repeated/prolonged usage might be cytotoxic to the cornea, especially the outmost human corneal epithelium (HCEP). The aim of the present study was to characterize the cytotoxicity of carteolol to HCEP and its underlying cellular and molecular mechanisms using an in vitro model of HCEP cells. After HCEP cells were treated with carteolol at concentrations varying from 2% to 0.015625%, the cytotoxicity, apoptosis-inducing effect and pro-apoptotic pathway was investigated, respectively. Our results showed that carteolol at concentrations above 0.03125% induced time- and dose-dependent growth retardation, cytopathic morphological changes and viability decline of HCEP cells. Moreover, carteolol induced G1 phase arrest, plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCEP cells. Furthermore, carteolol also induced activation of caspase-9 and −3, disruption of mitochondrial transmembrane potential, up-regulation the cytoplasmic amount of cytochrome c and apoptosis-inducing factor, and up-regulation of pro-apoptotic Bax and Bad, down-regulation of anti-apoptotic Bcl-2 and Bcl-xL. In conclusion, carteolol above 1/64 of its clinical therapeutic dosage has a time- and dose-dependent cytotoxicity to HCEP cells, which is achieved by inducing apoptosis via triggering Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway.

Nonsteroidal Anti-Inflammatory Drugs Cause Inhibition of the Growth Plate in Cultured Rat Metatarsal Bones: Retracted.

Little is known presently about the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the longitudinal bone growth occurring in the growth plate. We have examined the effects of administration of different types of NSAIDs on the longitudinal growth of the growth plate using a fetal rat metatarsal bone culture model. Cultured fetal rat metatarsal bones were used to study the effect of nonselective (indomethacin) and selective cyclooxygenase-2 (COX-2, celecoxib) NSAIDs on longitudinal bone growth. The effect of NSAIDs on proliferation and apoptosis of growth plate chondrocytes were analyzed by BrdU incorporation and a TUNEL assay. Prostaglandin E2 (PGE2) production was measured by an ELISA kit. We also examined the effect of exogenous PGE2 on the growth plate. NSAIDs caused a dose-dependent growth retardation of cultured metatarsal bones. Both nonselective and COX-2 selective NSAIDs inhibit longitudinal bone growth. We found that NSAIDs suppressed the proliferation of chondrocytes and production of PGE2, and increased the apoptosis of chondrocytes. Supplemental PGE2 could not reverse the effects of NSAIDs on the growth plate. Our data show that NSAIDs induce a dose-dependent growth retardation of cultured rat metatarsal bones. A detailed analysis revealed decreased proliferation and increased apoptosis of chondrocytes in the growth plate, indicating that COX-2 is responsible for PGE2 production in growth plate chondrocytes. The data from the present study suggest that deleterious effects on the growth plate by chronic NSAIDs use should be considered for children who have chronic inflammatory diseases, such as juvenile rheumatoid arthritis.

Abstract 2471: EGFRvIII T-cell TandAbs are specific and highly potent drug candidates for the treatment of a variety of solid tumors

Abstract To harness the cytotoxic capacity of immune effector cells for the treatment of several types of solid tumors, we developed tetravalent bifunctional antibodies that recognize EGFRvIII, the deletion variant III of EGFR. Their second functionality binds with high affinity to CD3, thereby directing T-cells to eliminate EGFRvIII+ cancer cells. The expression of EGFRvIII on various solid tumor types, and its absence from healthy tissues, provides an opportunity to develop cytotoxic antibodies that solely target cancer; these would spare normal tissues, and substantially reduce the side effects associated with EGFR therapy. Using phage display, we identified scFvs that selectively bind to EGFRvIII but not to EGFR. The binding affinities of these highly EGFRvIII-specific antibodies were substantially improved, employing affinity maturation techniques, and achieved KDs in the 100pM range and lower. We engineered a panel of bispecific EGFRvIII/CD3 TandAbs with a broad range of binding and cytotoxic properties. Mono- and bivalent binding, specificity for both EGFRvIII and CD3, T cell-mediated cytotoxic activity, and target-mediated T-cell activation were characterized in a panel of in vitro assays. EGFRvIII/CD3 TandAbs exhibit exquisite specificity towards the EGFRvIII antigen in Western Blot, SPR, ELISA, and FACS assays of EGFRvIII+ cells. No specific binding was observed to recombinant EGFR antigen or to EGFR-expressing cells. Apparent affinities of EGFRvIII/CD3 TandAbs to EGFRvIII were up to 25fold improved relative to the monovalently binding scFvs, and achieved a KD of 11pM for the best binding EGFRvIII/CD3 TandAb. Improvement of the binding affinities in the bivalent TandAb format was largely due to slower dissociation. TandAbs with high affinity for EGFRvIII were most potent in killing assays, displaying cytotoxicity towards EGFRvIII-expressing F98 glioma and CHO cells with EC50 in the range of 1pM-10pM. No cytotoxicity was observed on EGFR+ cells or EGFRvIII- cells up to the maximally-evaluated TandAb concentration of 0.5μM (which is 100000 fold higher than the EC50) demonstrating the high selectivity of EGFRvIII/CD3 TandAbs for the tumor-specific EGFRvIII. High affinity binding to CD3 was necessary for efficacious T cell recruitment as shown by the correlation of CD3-binding and cytotoxic potency of EGFRvIII/CD3 TandAbs. Importantly, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as demonstrated by their lack of proliferation: this specificity contributed to a good preclinical safety profile. Biophysical and pharmacological characterization of several candidates is currently ongoing, whereby the first EGFRvIII/CD3 TandAbs demonstrated a robust dose-dependent growth retardation of EGFRvIII+ subcutaneous xenograft tumors. In summary, EGFRvIII/CD3 TandAbs are specific and highly potent drug candidates for the treatment of EGFRvIII+ malignancies. Citation Format: Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Carmen Herbrecht, Stefan Knackmuss, Erich Rajkovic, Vera Molkenthin, Eugene A. Zhukovsky. EGFRvIII T-cell TandAbs are specific and highly potent drug candidates for the treatment of a variety of solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2015-2471

Abstract 2643: A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers

Abstract To harness the cytotoxic capacity of T cells for the treatment of EGFRvIII+ cancers, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 (or CD16A) and EGFRvIII, the EGFRvIII/CD3 (and EGFRvIII/CD16A) RECRUIT-TandAb. EGFR dysregulation has been linked to numerous cancers, and both small molecules and EGFR targeting antibodies have successfully reached the clinic. The deletion variant III (EGFRvIII), the most common mutant form of EGFR is expressed exclusively in cancer tissues contributing to oncogenic transformation. The restricted EGFRvIII expression on various solid tumor types provides an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues, and substantially reduce the side effects associated with EGFR therapy. Using phage display libraries, we identified scFvs that selectively bound to the mutated and not the native form of EGFR. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies; hence we engineered a panel of bispecific EGFRvIII/CD3 TandAbs, capable of T cell-recruitment, with a broad range of binding and cytotoxic properties. The TandAbs' binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. They exhibited exquisite specificity towards EGFRvIII in ELISA and FACS assays, with no wild-type EGFR binding observed, up to the maximally evaluated TandAb concentration of 1 μM. The most potent TandAbs displayed cytotoxicity towards EGFRvIII expressing F98 glioma and CHO cells with EC50 = 25 pM. We also assayed the cytotoxicity of these TandAbs towards EGFR+ cells as a more sensitive probe of residual binding to the native form. No cytotoxicity was observed up to the maximally evaluated TandAb concentration of 0.5 μM. The cytotoxic potency of the EGFRvIII/CD3 TandAb was higher than that of a comparator bivalent bispecific antibody, constructed from different anti-EGFRvIII/CD3 moieties or of EGFRvIII/CD16A TandAbs, which recruit NK cells instead of cytotoxic T cells. EGFRvIII/CD3 TandAbs with high affinity binding to CD3 were most efficacious for T cell recruitment and tumor cell killing and yet, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as measured by their lack of proliferation, contributing to a good preclinical safety profile. In vivo EGFRvIII/CD3 TandAbs demonstrated a robust dose-dependent growth retardation of EGFRvIII+ subcutaneous Xenograft tumors in NOD/scid mice reconstituted with human PBMC; this tumor growth inhibition was more pronounced than that associated with cetuximab. In summary, our data demonstrate the strict specificity and high potency of the anti-tumor cytotoxicity mediated by the EGFRvIII/CD3 TandAb, a novel highly potent drug candidate for the treatment of EGFRvIII+ malignancies. Citation Format: Eugene A. Zhukovsky, Kristina Ellwanger, Uwe Reusch, Markus Eser, Fionnuala McAleese, Ivica Fucek, Carmen Burkhardt, Stefan Knackmuss. A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2643. doi:10.1158/1538-7445.AM2014-2643

Bioaccumulation and behavioral effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in perinatally exposed mice

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants that have become pervasive environmental contaminants and may contribute to adverse health outcomes. We evaluated in mice the developmental neurotoxicity of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), one of the most abundant PBDE congeners detected in animal and human tissues. Female C57BL/6J mice were exposed to daily doses of 0, 0.03, 0.1 or 1 mg/kg beginning 4 weeks prior to conception, continuing through gestation and lactation, and ending at weaning on postnatal day (PND) 21. Levels of BDE-47 in blood, brain, liver and adipose tissues of dams were markedly increased after 4 weeks of exposure, around the time of mating, and continued to increase through the time of parturition. Blood levels of BDE-47 in the dosed dams were within the range reported in humans. BDE-47 tissue levels in the dams decreased between parturition and weaning, possibly reflecting mobilization during lactation. Brain BDE-47 levels in the offspring at PND 1 approached those of the dams at parturition. Perinatal exposure to BDE-47 resulted in significant dose dependent growth retardation, slower motor performance in several behavioral tests, and mice exposed to 1 mg/kg/day BDE-47 showed altered performance in the Morris water maze. There were no differences between groups in the numbers of pyramidal neurons in hippocampus CA1. These results document accumulation of BDE-47 in several organ systems following exposure to low-levels of BDE-47, and provide evidence that such exposure is associated with early behavioral deficits in exposed neonates.

Tamoxifen induces permanent growth arrest through selective induction of apoptosis in growth plate chondrocytes in cultured rat metatarsal bones

Estrogen affects skeletal growth and promotes growth plate fusion in humans. High doses of estrogen have been used to limit growth in girls with predicted extreme tall stature; a treatment which has been associated with severe side effects. Selective estrogen receptor modulators (SERMs) could potentially be used as an alternative treatment. We chose to study the effects of Tamoxifen (Tam), a first generation SERM that has been used in the treatment of pubertal gynecomastia or McCune–Albright syndrome. Cultured fetal rat metatarsal bones were used to study the effects of Tam on longitudinal bone growth. In sectioned bones, chondrocyte apoptosis and proliferation were analyzed by TUNEL assay and BrdU incorporation, respectively. We also used a human chondrocytic cell line, HSC-2/8, to study the effects of Tam on apoptosis (FACS analysis and Cell Death detection ELISA) and caspase activation (caspase substrate cleavage and Western immunoblotting). Tam caused a dose-dependent growth retardation of cultured metatarsal bones. No catch-up growth was observed after Tam was removed from the culture medium. Detailed analysis of sectioned growth plate cartilage revealed increased apoptosis of chondrocytes within the resting and hypertrophic zones. HCS-2/8 cells also underwent apoptosis upon Tam treatment. Tam-induced apoptosis was caspase-dependent and completely abrogated by either caspase-8 or -9 inhibitors. A substrate assay revealed that caspase-8 is first activated followed by caspase-9 and -3. Finally, FasL secretion was stimulated by Tam and blocking of either FasL or Fas decreased Tam-induced apoptosis in chondrocytes. We here describe a novel mechanism of tamoxifen-induced apoptosis in chondrocytes, involving the activation of caspases and the FasL/Fas pathway, which diminishes the potential for bone growth.

Maternal passive smoking during pregnancy and foetal developmental toxicity. Part 2: histological changes.

1. Evidence has been accumulating on the growth suppressing effects of maternal passive smoking on foetus. Reviewing all literature released during the last two decades and screening for all possible variables such as previous smoking history, maternal age and weight gain, parity and length of gestation, placental weight, and diet, we found no reason to doubt the role of passive smoking during pregnancy in the induction of growth retardation. However, no literature indicates whether these birthweight deficits might hint at other possible hidden abnormalities in tissues. To verify this question, we performed an experiment on rats. 2. We have already reported that pups born to rats with previous exposure to cigarette's sidestream smoke during pregnancy showed a significant and dose-dependent growth retardation. Those pregnant rats were exposed each in a 150 dm3 glass chamber to diluted sidestream smoke of either 1, 2, 3 or 4 commercial blond filter brand cigarettes during either first, second or third week of pregnancy. We have selected a part of each group of pups at random and examined for possible histological changes of lung, liver, stomach, kidney and intestinal tissues. 3. Compared to controls, lung tissues of newborns of smoke exposed mothers showed an enhanced incidence of apoptosis, mesenchymal changes, and hyperplasia of bronchial muscles. Pronounced abnormal changes in haematopoiesis and proliferation of bile duct cells were the most variations from norm observed in liver tissues of exposed pups. Immature glomeruli of kidney, epithelhypoplasia of stomach, and hypoplasia of intestinal villi were common among newborns of exposed mothers than among controls. 4. These results indicate that passive smoking upon pregnancy causes abnormal morphological changes in internal tissues of newborns. At present, we can draw no conclusion as to whether these histological changes will result in functional malformations or possibly late effects, although they should be expected.

A calcium agonist, Bay k 8644, suppresses the embryotoxic effects induced by dihydropyridines calcium channel blockers in cultured rat embryos.

Day 9 rat embryos were exposed to 1,4-dihydropyridine calcium channel blockers; nifedipine (NIF), nicardipine (NIC) or nitrendipine (NIT), for 48 hr in the whole embryo culture system. There were dose-dependent growth retardation and abnormalities, predominantly in cardiovascular system. The three compounds exhibited very similar pattern of dysmorphogenic effects, but the potency of these compounds were quantitatively different. The incidences of embryos with the abnormalities were 100%, 100% and 85% following either exposure of NIF, NIC or NIT at concentration of 300, 8 and 15 microM, respectively. This study was to investigate whether these blocker-induced embryotoxicity was due to calcium channel blocking properties themselves in the embryos. Day 9 rat embryos were co-exposed to 1,4-dihydropyridine calcium channel agonist, Bay k 8644 (BAY) and each calcium channel blocker under the same culture condition. The retarded embryonic growth induced by 200 or 300 microM of NIF, 8 microM of NIC and 15 microM of NIT nearly of completely ameliorated when embryos were co-exposed with BAY at one-third or half concentration of each calcium channel blocker. Supplementation of BAY reduced the incidence of abnormalities by NIF-, NIC- and NIT-alone. These results suggested that one of mechanisms for embryotoxicity induced by calcium channel blocker was directly related to channel blocking property of the chemicals.

Early events following maternal exposure to 5-fluorouracil lead to dysmorphology in cultured embryonic tissues.

The chemotherapeutic agent 5-fluorouracil (5-FU) is teratogenic in a number of species, yet the mechanism(s) of its developmental toxicity are not fully understood. Administration of 5-FU to the pregnant CD rat on day 14 of gestation results in dose-dependent growth retardation and numerous malformations in near-term fetuses, including hindlimb defects and cleft palate. Following treatment, a number of rapid biochemical and cellular alterations are detectable in embryonic hindlimbs, craniofacial and other tissues, which include thymidylate synthetase (TS) inhibition and altered cell cycle progression. In order to assess the importance of these early events in 5-FU-induced dysmorphogenesis, embryonic mid-facial tissues and hindlimbs were dissected 3 or 6 hr after administration of 5-FU to the dam and placed in explant culture. After 5 days in culture, craniofacial explants were evaluated morphologically for palatal closure and growth was assessed by measuring total protein and DNA content. Hindlimb explants were stained for cartilage using alcian blue to evaluate development of the digits. Craniofacial explants cultured at either 3 or 6 hr after exposure exhibited dose-dependent growth retardation and defects of palatal fusion at the end of the culture period. Deficits in protein and DNA content were similar to those in craniofacial tissues that continued to develop in utero after treatment, although morphological defects in cultured explants did not correlate well with the incidence of cleft palate in vivo. Dose-dependent deficits in metatarsal and phalanx development were observed in hindlimb explants dissected either 3 or 6 hr after maternal treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and adaptation of the green alga Chlamydomonas reinhardtii on diesel exhaust particle extracts

SUMMARY: We have examined the growth and photosynthesis of liquid cultures of the eukaryotic green alga Chlamydomonas reinhardtii incubated with iso-octane extracts of diesel particulate exhaust. At concentrations up to 0.125% (v/v), the extracts led to a dose-dependent growth retardation. Higher concentrations caused cell death. After a prolonged lag phase, air-grown illuminated algae, stressed with sublethal concentrations of diesel exhaust extracts, adapted to the pollutants. Further addition of toxic extract up to a concentration of 2.5 (v/v) affected neither growth nor photosynthesis. Media of adapted and non-adapted cells were analysed by HPLC and GC-MS and the concentrations of the remaining toxic compounds were determined. The results showed that the amounts of certain components decreased during incubation with adapted cells.

Experimental screening of BCG preparations produced for cancer immunotherapy: safety and immunostimulating and antitumor activity of four consecutively produced batches.

Four consecutively produced batches of Bacillus Calmette-Guérin (BCG) especially intended to be used for cancer immunotherapy were investigated for consistency of the vaccine. Each batch was investigated directly after production of the vaccine, so that the four batches were not tested simultaneously. The activity of the four batches was investigated in general safety assays, immunostimulation assays, and two different tumor models. General safety assays showed dose-dependent growth retardation and increased serum glutamic pyruvic transaminase activity in mice, and a long-lasting temperature rise in rabbits after IV administration of the BCG preparations. In a skin reactivity assay, reactions were found acceptable for all preparations when compared with a reference batch. The results of the immunostimulation and antitumor studies can be summarized as follows. All four batches induced a specific delayed-type hypersensitivity reaction to PPD, indicating the induction of cell-mediated immunity. A stimulating effect on lymphoreticular organs was concluded from increased spleen weight and enhanced cell proliferation in draining lymph nodes. Enhanced macrophage function (phagocytosis and killing of bacteria) was demonstrated by an increased resistance to Listeria monocytogenes. YAC lymphoma target cells were killed nonspecifically by BCG-activated peritoneal exudate cells (PEC), indicating the induction of natural killing activity by BCG. Intralesional injection of BCG induced tumor regression in the guinea pig line 10 hepatocellular carcinoma, followed by immunity to the line 10 tumor. In the murine 5D04 squamous cell carcinoma, BCG had no effect on the primary tumor. However, IV-injected BCG resulted in a decreased number of lung metastases. In general, the four consecutively produced batches showed similar safety and activity in the immunostimulation assays and antitumor activity. Since only minor differences between the batches were found, which can also be attributed to the variation in experimental conditions common to biological assays, it is concluded that the vaccine batches produced show an acceptable consistency.

Comparative 90-day toxicity of two decarboxylase inhibitors, benserazide and carbidopa, in the rat

The toxicity of two decarboxylase inhibitors, benserazide and carbidopa, was compared in a 90-day study in rats given doses from 32 to 260 mg/kg/day. No mortality directly caused by the drugs occurred. Late growth retardation in the high-dosage benserazide groups was apparently caused by difficulties in locomotion secondary to skeletal deformations. In the carbidopa groups, a uniform dose-dependent growth retardation was seen from the beginning. Hematological changes were not prominent. SGPT activities were decreased by both drugs after 6 weeks and more so after 12 weeks, presumably by direct inhibition. Effects on organ weights were difficult to interpret due to the serious growth retardation. No important histopathologic anomalies were seen, except in the skeleton of groups given the highest dosage of benserazide. A disturbance in enchondral bone formation caused secondary distortions in the skeleton impairing locomotion. Earlier results from separate studies suggesting a large difference between the two substances in toxicity for the rat could not be substantiated.

A new model for embryotoxicity testing: teratogenicity and pharmacokinetics of valproic acid following constant-rate administration in the mouse using human therapeutic drug and metabolite concentrations

The limitations of a conventional testing procedure for embryotoxicity-multiple dosing throughout the organogenesis period of the mouse or rat-are discussed for drugs such as valproic acid (VPA) which exhibit pharmacokinetic properties in these species which are very different from those in the human. Administration of VPA to the mouse, once each day, resulted in peak plasma drug concentrations 10 fold higher than human therapeutic plasma levels, after which the drug levels rapidly decreased to insignificant levels which persisted until the time of the next dose. Dose-dependent growth retardation, a sharp increase in the resorption rate and a high incidence of exencephaly were observed with this dosing regimen. A new mouse model was developed where constant-rate application of the drug with osmotic minipumps resulted in drug levels throughout the organogenesis period which were similar to those observed during human therapy. Such therapeutic drug levels produced a slight but significant fetal growth retardation and increased resorption rates, but not exencephaly. It is suggested that maintaining plasma concentrations in the experimental animal comparable to human therapeutic drug levels should offer a more realistic model for drug-embryotoxicity testing.

Liver cancer and precancerous changes in rats induced by the basic fraction of tryptophan pyrolysate

The basic fraction of a tryptophan pyrolysate (Trp-P-BF) was given orally to Wistar rats for about 2 years. In Experiment I, 5 male rats each were given 0.2, 0.4, 0.6 and 0.8% Trp-P-BF. Dose-dependent growth retardation was observed in these groups and neoplastic nodules were found in the liver of 1 rat given 0.2% Trp-P-BF and a hepatocellular carcinoma was found in 1 rat given 0.4% Trp-P-BF diet. In Experiment II, 25 rats of both sexes were fed 0.5% or 0.2% Trp-P-BF diet. Neoplastic nodules were induced in 2 of 22 males and 5 of 18 females given 0.2% Trp-P-BF diet. Mammary adenomas developed, but no neoplastic nodules were found in the liver of rats fed on 0.05% Trp-P-BF or control diet. Females were more sensitive to Trp-P-BF than males.