Comparison of Phototoxicity Sensitivity by the Neutral Red Uptake Method for BALB/c 3T3, HaCaT, and HDFa Cells In Vitro

ABSTRACT Introduction Phototoxicity is an acute photoinduced reaction. The 3T3 neutral red uptake (NRU) phototoxicity test has high sensitivity for the determination of phototoxic substances. To further optimize the method, this study mainly focused on comparing the phototoxicity sensitivity by using the NRU method for BALB/c 3T3, HaCaT, and HDFa cells in vitro. Methods The NRU method was used to evaluate the phototoxicity of chlorpromazine hydrochloride (CPZ), amiodarone hydrochloride (Amiodar), and L-histidine (L-His) on BALB/c 3T3 cells, HaCaT cells, and HDFa cells. The sensitivity of different cells to ultraviolet (UVA) irradiation in vitro was studied. Results L-His showed no phototoxicity, but the phototoxicity of CPZ and Amiodar showed different sensitivities among the three kinds of cells. The in vitro phototoxicity evaluation of HaCaT cells is closer to that of primary human fibroblasts. Conclusion This study provides a reference for cell line selection to optimize the existing in vitro evaluation method of 3T3 NRU phototoxicity.

Posttreatment Strategy Against Hypoxia and Ischemia Based on Selective Targeting of Nonnuclear Estrogen Receptors with PaPE-1

Newly synthesized Pathway Preferential Estrogen-1 (PaPE-1) selectively activates membrane estrogen receptors (mERs), namely, mERα and mERβ, and has been shown to evoke neuroprotection; however, its effectiveness in protecting brain tissue against hypoxia and ischemia has not been verified in a posttreatment paradigm. This is the first study showing that a 6-h delayed posttreatment with PaPE-1 inhibited hypoxia/ischemia-induced neuronal death, as indicated by neutral red uptake in mouse primary cell cultures in vitro. The effect was accompanied by substantial decreases in neurotoxicity and neurodegeneration in terms of LDH release and Fluoro-Jade C staining of damaged cells, respectively. The mechanisms of the neuroprotective action of PaPE-1 also involved apoptosis inhibition demonstrated by normalization of both mitochondrial membrane potential and expression levels of apoptosis-related genes and proteins such as Fas, Fasl, Bcl2, FAS, FASL, BCL2, BAX, and GSK3β. Furthermore, PaPE-1-evoked neuroprotection was mediated through a reduction in ROS formation and restoration of cellular metabolic activity that had become dysregulated due to hypoxia and ischemia. These data provide evidence that targeting membrane non-GPER estrogen receptors with PaPE-1 is an effective therapy that protects brain neurons from hypoxic/ischemic damage, even when applied with a 6-h delay from injury onset.

Biological Innocuousness of a Device developed to select the Optimum Spermatozoa to be used in the Treatment of Infertility

The sperm selection assay (SSA) is a method based on chemotaxis to obtain spermatozoa at the optimum physiological state to successfully fertilize the egg. It consists of a device made of acrylic and an attractant solution which includes progesterone. We evaluate potential cytotoxicity interactions by means of Neutral Red uptake, the MTT and colony formation assays, according to ISO normative. Here we showed that even stressing the conditions of the assays, the SSA device alone or together with the progesterone solutions employed, showed to be innocuous for the cells. Suggesting that SSA could be incorporated into the ART procedures.

Differential Effects of the Flavonolignans Silybin, Silychristin and 2,3-Dehydrosilybin on Mesocestoides vogae Larvae (Cestoda) under Hypoxic and Aerobic In Vitro Conditions

Mesocestoides vogae larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans—silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)—on M. vogae larvae at concentrations of 5 and 50 μM under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 μM, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 μM concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism.

Advances in acute toxicity testing: strengths, weaknesses and regulatory acceptance

Safety assessment of chemicals, pharmaceuticals, food and food ingredients, cosmetics, industrial products is very crucial prior to their approval for human uses. Since the commencement of toxicity testing (about 500 years ago, since 1520), significant advances have been made with respect to the 3Rs (reduction, refinement and replacement) alternative approaches. This review is focused on the update in acute systemic toxicity testing of chemicals. Merits and demerits of these advances were also highlighted. Traditional LD50 test methods are being suspended while new methods are developed and endorsed by the regulatory body. Based on the refinement and reduction approaches, the regulatory body has approved fixed dose procedure (FDP), acute toxic class (ATC) method and up and down procedure (UDP) which involves few numbers of animals. In terms of replacement approach, the regulatory body approved 3T3 neutral red uptake (NRU), the normal human keratinocyte (NHK), and the 3T3 neutral red uptake (NRU) phototoxicity test for acute phototoxicity. However, other promising replacement alternatives such as organ on chip seeded with human cells for acute systemic toxicity and 3T3 neutral red uptake (NRU) cytotoxicity test for identifying substances not requiring classification, as well as the in silico approaches are yet to receive regulatory approval. With this backdrop, a collaborative effort is required from the academia, industries, regulatory agencies, government and scientific organizations to ensure speedily regulatory approval of the prospective alternatives highlighted.

Antiangiogenic Activity and Cytotoxicity of Triterpenoids and Homoisoflavonoids from Massonia pustulata and Massonia bifolia

The Hyacinthaceae family (sensu APGII), with approximately 900 species in around 70 genera, plays a significant role in traditional medicine in Africa as well as across Europe and the Middle and Far East. The dichloromethane extract of the bulbs of Massonia pustulata (Hyacinthaceae sensu APGII) yielded two known homoisoflavonoids, (R)-5-hydroxy-3-(4-hydroxybenzyl)-7-methoxy-4-chromanone 1 and 5-hydroxy-3-(4-hydroxybenzyl)-7-methoxy-4-chromone 2 and four spirocyclic nortriterpenoids, eucosterol 3, 28-hydroxyeucosterol 4 and two previously unreported triterpenoid derivatives, (17S,23S)-17α,23-epoxy-3β,22β,29-trihydroxylanost-8-en-27,23-olide 5, and (17S, 23S)-17α,23-epoxy-28,29-dihydroxylanost-8-en-3-on-27,23-olide 6. Compounds 1, 2, 3, and 5 were assessed for cytotoxicity against CaCo-2 cells using a neutral red uptake assay. Compounds 1, 2, and 5 reduced cell viability by 70% at concentrations of 30, 100, and 100 µM, respectively. Massonia bifolia yielded three known homoisoflavonoids, (R)-(4′-hydroxy)-5-hydroxy-7-methoxy-4-chromanone 1, (R)-(4′-hydroxy)-5,7-dihydroxy-4-chromanone 7 and (R)-(3′-hydroxy-4′-methoxy)-5,7-dihydroxy-4-chromanone 9, two previously unreported homoisoflavonoids, (E)-3-benzylidene-(3′,4′-dihydroxy)-5-hydroxy-7-methoxy-4-chromanone 8 and (R)-(3′,4′-dihydroxy)-5-hydroxy-7-methoxy-4-chromanone 10, and a spirocyclic nortriterpenoid, 15-deoxoeucosterol 11. Compounds 1, 1Ac, 7, 8, 9, and 10 were screened for antiangiogenic activity against human retinal microvascular endothelial cells. Some compounds showed dose-dependent antiproliferative activity and blocked endothelial tube formation, suggestive of antiangiogenic activity.