Ultraviolet B radiation affects epithelial cell morphology and ultrastructure in the hepatopancreas of the freshwater decapod Macrobrachium olfersii.

The hepatopancreas is the digestive organ of crustaceans, and plays important roles also in the synthesis and secretion of sexual hormones, immunological defenses and xenobiotic detoxification. Although the importance of this organ in crustaceans cannot be underestimated, the effects of ultraviolet B (UVB) radiation on hepatopancreas are poorly understood. Moreover, Macrobrachium prawns, have a transparent carapace, which make them more susceptible to UVB radiation, since their internal organs, such as hepatopancreas, are easily reached by solar radiation. Therefore, we aimed to evaluate UVB radiation toxicity on the morphology and morphometry of hepatopancreatic epithelial cells, and to investigate these UVB effects in subcellular compartments of the ecologically-important freshwater decapod, Macrobrachium olfersii. Hepatopancreas from the UVB-irradiated group showed a granular cytoplasm, with non-defined cell limits. Morphometric analyses revealed that the UVB-irradiated group exhibited a higher frequency of fibrillar (F-cell), resorptive (R-cell) and midget (M-cell), and decreased the blister-like (B-cell). It was also observed increased vacuole frequencies and increased F-, B- and R-cell volumes in the UVB-irradiated group. In addition, it was observed increased B-cell vacuolar volumes and decreased R-cell vacuolar volumes. Ultrastructural alterations occurred in subcellular compartments in F- and R-cells, e.g. loss of mitochondrial crests, morphologically compatible with mitochondrial fission, rough endoplasmic reticulum cisternae dilation, dilation of Golgi lamellar sacs, and increased vacuole and concentric membrane formation in the UVB-irradiated group. Our data showed that the hepatopancreas is an important target of UVB radiation, as demonstrated by a series of organ-specific morphological and morphometric impairments. Therefore, cell damage caused by UVB radiation can compromise metabolic functions in epithelial cells from the hepatopancreas, potentially affecting absorption, secretion and digestion processes, vitellogenin synthesis, immune responses and xenobiotic detoxification.

Impact of homocysteine on vasculogenic factors and bone formation in chicken embryos.

Developmental endochondral ossification requires constant blood supply, which is provided by the embryonic vascular network. High levels of homocysteine (Hcy) have vasculotoxic properties, but it remains unclear how Hcy disrupts blood vessel formation in endochondral ossification. Thus, we investigated the toxicity of Hcy on contents of vasculogenic factors (VEGF, VCAM-1, NOS3) and osteocalcin, using developing limbs as model. Chicken embryos were submitted to treatment with 20 µmol D-L Hcy at 12H&H and the analyses occur at 29H&H and 36H&H. We did not identify differences in the area of limb ossification in Hcy-treated (7.5 × 105 µm2 ± 3.9 × 104) and untreated embryos (7.6 × 105 µm2 ± 3.3 × 104) at 36H&H. In Hcy-treated embryos, we observed a significantly decrease of 46.8% at 29H&H and 26.0% at 36H&H in the number of VEGF-reactive cells. Also, treated embryos showed decrease of 98.7% in VCAM-1-reactive cells at 29H&H and 34.6% at 36H&H. The number of NOS3-reactive cells was reduced 54.0% at 29H&H and 91.5% at 36H&H, in the limbs of Hcy-treated embryos. Finally, in Hcy-treated embryos at 36H&H, we observed a reduction of 58.86% in the number of osteocalcin-reactive cells. Here, we demonstrated for the first time that the toxicity of Hcy is associated with a reduction in the contents of proteins involved in blood vessel formation and bone mineralization, which interferes with endochondral ossification of the limb during embryonic development. Graphical abstract.

Effect of UVB radiation exposure in the expression of genes and proteins related to apoptosis in freshwater prawn embryos.

Our previous studies showed that embryos of the freshwater prawn Macrobrachium olfersii exposed to ultraviolet B (UVB) radiation exhibited DNA damage, excessive ROS production, mitochondrial dysfunction and increased hsp70 expression, which are able, independently or together, to induce apoptosis. Thus, we attempted to elucidate some key apoptosis-related genes (ARG) and apoptosis-related proteins (ARP) and their expression during different stages of embryonic development, as well as to characterize the chronology of ARG expression and ARP contents after UVB radiation insult. We demonstrate that p53, Bax and Caspase3 genes are active in the embryonic cells at early embryonic developmental stages, and that the Bcl2 gene is active from the mid-embryonic stage. After UVB radiation exposure, we found an increase in ARP such as p53 and Bak after 3h of exposure. Moreover, an increase in ARG transcript levels for p53, Bax, Bcl2 and Caspase3 was observed at 6h after UVB exposure. Then, after 12h of UVB radiation exposure, an increase in Caspase3 gene expression and protein was observed, concomitantly with an increased number of apoptotic cells. Our data reveal that ARG and ARP are developmentally regulated in embryonic cells of M. olfersii and that UVB radiation causes apoptosis after 12h of exposure. Overall, we demonstrate that embryonic cells of M. olfersii are able to active the cell machinery against environmental changes, such as increased incidence of UVB radiation in aquatic ecosystems.

Homocysteine-induced changes in cell proliferation and differentiation in the chick embryo spinal cord: implications for mechanisms of neural tube defects (NTD).

Maternal hyperhomocysteinemia during pregnancy is associated with increased risk of NTD in the offspring. Our study investigated the effects of homocysteine (Hcy) on proliferation and neuronal differentiation of the spinal cord cells in a chick embryo model. Embryos were treated with 20µmol D-L Hcy/50µL saline solution at embryonic day 2 (E2) and analyzed at embryonic days 4 (E4) and 6 (E6). Control embryos received exclusively 50µL saline solution. We performed immunolocalization and flow cytometry analyses using antibodies anti-phosphohistone H3 (pH3), anti-proliferating cell nuclear antigen (PCNA), anti-ß-tubulin III and anti-p53. Our results revealed that Hcy interferes in the proliferation of the neural cells, and that this effect is age-dependent and differed between Hcy-treated embryos with and without NTD. Also, Hcy induced a decrease of neuronal differentiation in the spinal cord at both embryonic ages. These findings contribute to clarifying the cellular bases of NTD genesis, under experimental hiperhomocysteinemia.

Developmental effects of exposure to ultraviolet B radiation on the freshwater prawn Macrobrachium olfersi: Mitochondria as a target of environmental UVB radiation.

In South America, increased UVB radiation has become an important environmental issue that is potentially threatening aquatic ecosystems. Considering that species exhibit different degrees of sensitivity to UVB radiation and that embryos are more sensitive than organisms at later life stages, the aim of this study was to characterize the effects of UVB radiation on subcellular compartments of embryos of the freshwater prawn Macrobrachium olfersi. This species lives and reproduces in clear and shallow waters, where UV radiation can fully penetrates. Embryos were irradiated with a UVB 6W lamp for 30min and examined after 1h, 12h, 24h and 48h of exposure. The irradiance of the UVB used simulates the UV radiation that embryos receive in the natural environment. The subcellular compartment most affected by the UVB radiation was the mitochondria, which exhibited a circular shape, a decrease in mitochondrial cristae, rupture of membranes and a morphology compatible with fission. These impairments were observed simultaneously with increased ROS production, just after 1h of UVB exposure. Thus, we investigated proteins related to mitochondrial fission (Drp-1) and fusion (Mfn-1), which are essential to cell maintenance. We found a significant increase in Drp-1 expression at all analyzed time-points and a significant decrease in Mfn-1 expression only after 24h of UVB exposure. Additionally, a decrease in embryonic cell viability was verified via the mitochondrial integrity assay. To conclude, we observed important mitochondrial dysfunctions against the environmental stress caused by UVB radiation. Moreover, the cellular responses found are critical and should not be disregarded, because they impact embryos that can potentially compromise the aquatic ecosystems.

Effects of homocysteine on mesenchymal cell proliferation and differentiation during chondrogenesis on limb development.

High levels of homocysteine (Hcy) are related to an increased risk of the occurrence of congenital anomalies, including limb defects. However, few evaluations about how toxic levels of Hcy affect limb development have been reported. We investigated whether Hcy can affect the cell cycle proteins and proteins involved in mesenchymal cell differentiation during limb development, in a chicken embryo model. Embryos were treated with 20 µmol d-l Hcy/50 µl saline at embryonic day 2 and analyzed at embryonic day 6. Untreated control embryos received exclusively 50 µl saline solution. To identify cells in proliferation and cell cycle proteins, as well as Pax1/9 and Sox9 proteins, we performed immunolocalization and flow cytometry analyses using the antibodies anti-phosphohistone H3, anti-p53, anti-p21, anti-proliferating cell nuclear antigen, anti-Pax1, anti-Pax9 and anti-Sox9. No significant differences in cell proliferation were observed between Hcy-treated and untreated embryos. We observed a decrease of the proliferating cell nuclear antigen and p21 proteins, both involved in the G1 phase of cell cycle progression. On the other hand, in mesenchymal cells of the limbs, Hcy induces an increase of p53 protein, which can be activated by DNA damage. In cell differentiation, Hcy induced an increase mainly of Pax9 and Sox9 proteins. Our data indicate that the treatment with Hcy changes the mesenchymal cell dynamics during limb development, but does not change the morphology of the cartilage molds. These findings provide information to understand better the cellular basis of the toxicity of Hcy on chondrogenesis during limb development.

Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme.

BACKGROUND: Neural tube defects (NTD) involve disruptions in the axial mesenchyme, and are related to an imbalance between folic acid (FA) and homocysteine (Hcy). This study evaluated the effects of FA/Hcy imbalance on cell proliferation and expression of the Pax 1/9 and Sox 9 gene products in the axial mesenchyme of chickens. METHODS: Embryos were incubated (38°C) and pretreated at 24 h and treated at 46 h of incubation. The experimental groups were: FA-pretreated with saline and treated with 0.5 µg FA/saline; Hcy-pretreated with 50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; FA+Hcy-pretreated with 0.5 µg FA/50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; and the control embryos were pretreated and treated with saline. Embryos were analyzed at E4 and E6. Immunohistochemistry was performed to identify proliferating cells and the expression of the gene products of Pax 1/9 and Sox 9. Total RNA of the E4 embryos was extracted and a RT-qPCR assay was performed to quantify Pax 1/9 mRNA expression. RESULTS: Hcy treatment caused spinal NTD and abnormalities in axial mesenchyme development, affecting the distribution of sclerotomal cells and chondrification. Hcy also reduced cell proliferation and changed the expression of Pax 1/9 and Sox 9 in the mesenchyme. CONCLUSIONS: Our data clarified the relationship between spinal NTD genesis and disruptions of Pax 1/9 and Sox 9 gene products in the axial mesenchyme caused by the FA/Hcy imbalance.

A novel monoclonal antibody that binds to hemocytes from shrimps and oysters.

The monoclonal antibody (MAb) LITO-1 was produced from a stable hybridoma cell line generated by the fusion of NS1 myeloma cells with spleen cells isolated from Balb/c mice immunized with a paraformaldehyde-fixed hemocyte suspension of Litopenaeus vannamei. This MAb reacted with all three hemocyte subtypes, but no reaction was observed with components of plasma. Immunohistochemistry assays demonstrated that LITO-1 was very effective in specifically distinguishing hemocytes infiltrated in several tissues such as striated muscle, brain, and hepatopancreas. Moreover, this antibody was able to recognize hemocytes from two shrimp species, Litopenaeus schmitti and Farfantepenaeus paulensis, as well as hemocytes of the oyster Crassostrea gigas. No reaction was observed against hemocytes from the terrestrial insect Triatoma klugi or with mammalian RAW cells. This novel MAb can be useful in revealing the presence and function of a conservative epitope in hemocytes of marine crustaceans and mollusks.

Effects of environmental and artificial UV-B radiation on freshwater prawn Macrobrachium olfersi embryos.

The recent decrease of the stratospheric ozone has resulted in an increase of ultraviolet-B (UV-B) radiation reaching the Earth's surface. In freshwater ecosystems with transparent water, UV-B rays easily penetrate and potentially cause harmful effects to organisms. In this study, embryos of the prawn Macrobrachium olfersi were used to evaluate the impact of UV-B rays in freshwater environments. We observed three groups of embryos: the first was to assess whether UV-B radiation produced morphological defects and/or biochemical impairments in the laboratory. The second was to check whether embryos with the same impairments as those observed in the laboratory were found in their environment, under natural solar radiation. The third group was the non-irradiated control. The embryos irradiated with 310 mW cm(-2) UV-B for 30 min showed morphological alterations similar to those observed in embryos from the environmental control group. The most important effects of the UV-B radiation observed in M. olfersi embryos were morphological (1.2% of the total number of embryos from the environment and 2.8% of the total number of irradiated embryos), pigmentation changes in the eyes (78.0% of the total number of embryos from the environment and 98.9% of the total number of irradiated embryos), and disruption of the chromatophores (46.9% of the total number of embryos from the environment and 95.5% of the total number of irradiated embryos). We also observed an increase in egg volume, which was accompanied by a significant increase in water content in UV-B irradiated groups when compared with aquaria control embryos. In addition, a significant decrease in the mitotic index in eggs exposed to UV-B radiation was detected (0.17 for the embryos from the aquaria control, 0.10 for the embryos of the environmental control, and 0.04 for the irradiated groups). The low levels of NPSH and high levels of TBARS indicated that UV-B rays directly compromised the antioxidant function of the embryonic cells, leading to oxidative stress. Our combined morphological and biochemical analyses revealed important effects induced by UV-B on M. olfersi embryos, and the results suggest that the recent changes in global conditions may have injurious effects, at least on the embryos of freshwater prawns.