Fucoidan, a sulfated polysaccharide found abundantly in brown algae such as mozuku seaweeds, is classified as a water-soluble dietary fiber. Fucoidan reportedly exerts antitumor, anti-inflammatory, immunomodulatory, antioxidant, whitening, antibacterial, and antiviral activities. Owing to these properties, fucoidan has been widely used as an active ingredient for skin care purposes, including quasi-drug serums and hair growth products. However, fucoidan effects differ depending on the algal species, molecular weight, and other factors. Thus, it is crucial to thoroughly test fucoidan preparations and determine their suitability for individual applications. In this study, we probed the effects of mozuku-derived ultra-high-molecular-weight fucoidan (1700 kDa, normally ~300 kDa) preparations on indigenous skin bacteria and general bacteria. We observed that fucoidan preparations derived from Tongan mozuku Cladosiphon novae-caledoniae and Amami mozuku Cladosiphon okamuranus inhibited the growth of skin bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis. However, no growth inhibition was observed for the skin bacterium Cutibacterium acnes or general bacteria, such as Lactobacillus casei, Escherichia coli, and Bacillus subtilis. Given its ability to hinder the growth of skin disease-causing bacteria, mozuku-derived ultra-high-molecular-weight fucoidan holds promise for future applications in the pharmaceutical and cosmetic industries.

Flunitrazepam, a highly potent benzodiazepines, has a wide safety margin and widely used for insomnia treatment. However, a number of fatal poisoning cases involving a combination of flunitrazepam and other drugs have been reported. In instances of drug overdose deaths involving flunitrazepam in Japan, antipsychotic drugs like chlorpromazine are frequently used concomitantly. This study seeks to elucidate the pharmacokinetic interactions during the overdose of concurrent drugs, with a specific focus on the toxic effects of elevated doses of flunitrazepam and chlorpromazine in mice. Male ICR mice were intraperitoneally administered chlorpromazine (90 mg/kg) and flunitrazepam (200 mg/kg) either alone or concurrently. Body temperature was measured up to 24 hr after administration, and the number of deaths within 24 hr was quantified for each group. Additionally, flunitrazepam, chlorpromazine, and its active metabolite 7-hydroxychlorpromazine concentrations in serum and brain extracellular fluid were measured up to 24 hr after administration. Flunitrazepam enhanced the hypothermic effect of chlorpromazine, and acute intoxication deaths occurred only in the combination group. Cmax and AUC_0-24h of flunitrazepam in serum and brain were not affected by concomitant administration with chlorpromazine. While flunitrazepam significantly increased the AUC_0-24h of chlorpromazine, and the Cmax and AUC_0-24h of 7- hydroxychlorpromazine in serum. Flunitrazepam was shown to alter the toxicokinetics of chlorpromazine when administered in combination, thereby augmenting the toxicity of chlorpromazine and leading to lethal drug intoxication. This study underscores the significance of comprehending toxicokinetics not only for individual agents but also when utilized in combination.

Trifluoroacetic acid (TFA) has been used for production of pharmaceuticals including recently emerging middle size peptides. TFA has severe local toxicity like skin and respiratory tract irritation due to the strong acidity. Because almost all pharmaceutical products are prepared at neutral pH, derivation of permitted daily exposure (PDE) of TFA from the information with neutralized form could provide useful information on the risk management for protection of patients and production of pharmaceuticals. Sodium TFA caused hepatotoxicity in a rat 90-day. Based on the results of the 90-day study, we respectively proposed PDE for oral, parenteral and inhalation routes as 1.7, 0.005 and 0.84 mg/person/day. The derivation of PDE was done according to the concept described in ICH Q3C(R8) and Q3D(R2).

p-tert-butyl phenol (ptBP) is an alkylphenol organic compound that is used in the production of polycarbonates, phenolic resins, epoxy resins, etc., as a polymer chain terminator. However, it can occasionally be an impurity contaminant during the chemical synthesis of active pharmaceutical ingredients. Some alkylphenols are known as carcinogens or endocrine modulators, therefore controlling the impurity level to below the permitted daily exposure (PDE) value based on the toxicological risk assessment of ptBP should be considered. Here, we propose the PDE using toxicological information primarily obtained from OECD Existing Chemicals Database. Four scenarios for the oral PDE calculation were investigated, and the lowest value, 2 mg/day, was proposed as the most appropriate control criterion of ptBP based on the depigmentation toxicity seen in black mice. The PDE for the parenteral route was concluded to be 1 mg/day by applying a default bioavailability of 50% per ICH Q3D guideline.

The amidine compound 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is a strong organic base, is non-nucleophilic, and has been widely used in the organic synthesis of pharmaceuticals as a base catalyst. In the present study, we propose the permitted daily exposure (PDE) values of DBU by the oral, parenteral, and inhalation routes based on toxicological information in animals from the database of the European Chemicals Agency (ECHA). DBU exhibited mainly stomach toxicity caused by the corrosive effect of this strong alkali in rats. Although it is unlikely that stomach toxicity is induced in patients by DBU contamination of pharmaceuticals because most pharmaceutical products are adjusted to a near neutral pH, we calculated the PDE values conservatively based on the dosage at which no stomach-related findings were observed because no other noteworthy findings caused by systemic exposure were included in toxicity studies. By applying adjustment factors to the no-observed-adverse-effect-level (NOAEL) (120 mg/kg/day) in animal studies during the longest treatment period (3 months), we estimated the PDE values for oral, parenteral, and inhalation routes as 24, 12, and 6 mg/day, respectively. Derivation of the PDE was performed according to the concepts described by the International Conference on Harmonisation (ICH) Q3C(R8) and Q3D(R2) guidelines.

Covalent binding of chemicals to skin proteins represents the Molecular Initiating Event (MIE) of the skin sensitization process. We attempted to construct in silico models for predicting the reactivities of chemicals to cysteine measured by the Direct Peptide Reactivity Assay (DPRA) as a screening tool for skin sensitization potential of chemicals since there was no readily available in silico prediction model for reactivity classes of the DPRA. We used a dataset of 211 chemicals compiled based on the chemical reactivity to cysteine in the DPRA for model construction, and each chemical was classified as “Minimal-Low” or “Moderate-High” reactivity according to the percent cysteine depletion value in the DPRA. We constructed two independent classification models using two machine learning algorithms named Random Forest (RF) and Graph Convolutional Network (GCN), and a consensus model adopting prediction results when both of the GCN-based and the RF-based models were matched was also constructed. Performance evaluation showed that the RF-based model showed higher specificity than the GCN-based model and the GCN-based model showed higher sensitivity than the RF-based model. The consensus model showed high accuracy and high specificity of over 0.9. Comparison of the reactivity class predicted by the consensus model and the skin sensitization potential for humans revealed that all chemicals classified into the “Moderate-High” class were human skin sensitizers. In conclusion, the consensus model we constructed here may be a promising in silico screening tool to predict cysteine reactivity measured by the DPRA and skin sensitization potential of chemicals.

K16ApoE is a brain-targeted drug carrier that can deliver drugs throughout the brain and maintain sustained activity during delivery. However, related studies have revealed that K16ApoE has acute and high toxicity, and its toxicological mechanism remains unknown. Our previous experiments found that K16ApoE affects the blood state of mice. Thus, we performed a hemolysis assay to investigate whether K16ApoE could induce hemolysis, but we found that K16ApoE was unable to lyse red blood cells at lethal doses. Then, we monitored cerebral blood flow and perfusion after the mice received incremental doses of K16ApoE and found that K16ApoE could suddenly interrupt the blood flow and reduce the perfusion in the brain. K16ApoE could also disrupt the consistency of cerebral perfusion. Last, we examined the blood cells under the microscope and found that K16ApoE caused the cells to clump in the plasma and increase blood viscosity. Based on these findings, we confirmed that the toxicity of K16ApoE is mainly in the blood. The mechanism is that K16ApoE induces blood cell aggregation and interrupts blood flow.

Clarifying the cytochrome P450s (CYPs) changes in non-alcoholic fatty liver disease (NAFLD) is important for optimizing drug therapy. This study compared the expression of CYP isoforms in rat models of nonalcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). An NAFL model without tissue damage or inflammation was established by feeding rats a high-fat diet (HFD) for a relatively short period of 4 weeks. Feeding rats with a methionine-choline-deficient diet (MCDD) for 4 weeks produced steatohepatitis-like NASH. Here, the mRNA and protein expression levels of several CYP enzymes in NAFL and NASH models were compared with those in rats fed a control diet (CD). CYP1A2 expression and activity were upregulated in the NAFL model and downregulated in the NASH model, suggesting a reversal of CYP1A2 expression between NAFL and NASH. Differential expression of CYP1A2 in the NAFL and NASH models was observed in primary rat hepatocytes. These findings suggest that CYP1A2 expression/activity vary from the early stages of NAFL to NASH and that monitoring the pharmacokinetics of CYP1A2-metabolized drugs in humans with NAFL and NASH is necessary.

Various nanomaterials are used as food additives, medicines, and cosmetics; however, their biological effects are not completely understood. Since the cell membrane is the first point of contact between nanomaterials and cells, we investigated whether amorphous silica particles and titanium dioxide nanoparticles (nTiO₂) interact with the transporters present in the cell membrane. In this study, we prepared an adriamycin (ADM) resistant variant of the mouse lymphocytic leukemia cell line L1210 (L1210/ADM). Since our L1210/ADM cells were confirmed to excrete ADM in a P-glycoprotein (P-gp)-dependent manner, we investigated whether nano- and micro-sized amorphous silica particles (nSP and mSP) and nTiO₂ inhibited ADM excretion from L1210/ADM cells. Both nSP and nTiO₂ inhibited ADM efflux in a dose-dependent manner; however, mSP did not inhibit ADM efflux even at the highest dose (0.25 mg/mL). These results suggest that nSP and nTiO₂ interfere with P-gp, which is involved in ADM transport, and L1210/ADM cells are suitable for safety screening tests of nanomaterials.

In contrast with standard systemic drugs, the toxicity study of ophthalmic drug has unique design and packaging characteristics. The present survey aimed to characterize the nonclinical toxicological strategy of intravitreal administration (IVT) drugs by summarizing the toxicity study packages and comparing toxicity findings with clinical side effects. Safety pharmacology studies, toxicity studies, and clinical adverse reactions of the following seven IVT drugs were surveyed: pegaptanib sodium, ranibizumab, aflibercept, brolucizumab, faricimab, triamcinolone acetonide, and ranibizumab biosimilar. The toxicity study packages for IVT drugs were constructed according to ICH guidelines, although there were several differences between the modalities, application categories, and drugs. The characteristics of toxicity study packages include the fact that many safety pharmacology and acute toxicity endpoints need not be conducted as separate studies owing to the low systemic exposure. In addition, local toxicity findings (especially intraocular inflammation) require caution due to the relatively invasive administration method necessitates, and monkeys were mainly used as animal species in the IVT studies. Notably, certain drugs were found to have the severe adverse reactions of retinal vascular lesions, probably owing to immunoreaction. Importantly, these immunoreaction-related adverse reactions were not detected in these nonclinical toxicity studies. Therefore, risk assessments using toxicity studies, immunogenicity, and host impurities are important for the prediction and control of adverse reactions. This survey provides valuable information for the construction of a toxicity study design for IVT drug development.

Proliferating cells, such as tumor cells, require nucleotides for DNA replication. Mammalian cells are equipped with de novo purine and pyrimidine nucleotide biosynthesis pathways, and a salvage pathway that recycles purine bases, to supply nucleotides for the same. To avoid imbalance in intracellular nucleotide levels, de novo nucleotide biosynthesis pathway is regulated by feedback mechanisms, such as synthase inhibition by nucleotide products. Recently, we reported that the aldehyde dehydrogenase 1 family member L1 (ALDH1L1) consumes 10-formyltetrahydrofolate (10-fTHF), which is utilized by the de novo purine nucleotide synthesis, and results in the accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) during purine biosynthesis. Given that ZMP inhibits pyrimidine nucleotide synthesis, in the present study, we examined the effects of ZMP using brequinar, a dihydroorotate dehydrogenase inhibitor. ALDH1L1-mediated ZMP accumulation was unaffected by brequinar, and no effect was observed when brequinar was combined with 5-aminoimidazole-4-carboxamide riboside (AICAr), a nucleoside of ZMP. Furthermore, we examined involvement in the salvage pathway, because attenuation of de novo purine nucleotide synthesis may require a supply of purine nucleotides from the salvage pathway. The guanine analog, 6-thioguanine, and the 2'-deoxycytidine analog, cytarabine, were used in the assessment of dependency on the salvage pathway. We found that neither ALDH1L1-mediated ZMP accumulation nor the presence of AICAr affected the salvage pathway. Collectively, these results suggest that these purine and pyrimidine analogs can be useful for the treatment of tumor cells, regardless of ALDH1L1 expression.

Coffee which is one of the most popular beverages is under great attention because its attractive pharmacological effects such as anti-cancer properties, antioxidant and cell protective effects. Coffea arabica metabolites like cafestol and kahweol belonging to class of diterpene are expected to be as biotherapeutic drugs for maintaining human health via preventing serious illnesses. However, the understanding of the effects of these two diterpenes against human leukemia cells is still poor. In this study, we investigated the influences of these two coffee diterpenes on the viability and the all-trans retinoic acid (ATRA)-induced superoxide anion (O₂^-)-generating ability of human leukemia U937 cells. Cafestol and kahweol reduced cell viability at a concentration of 50 μM. In addition, 1 μM ATRA significantly reduced viability with cafestol at 48 hr and kahweol at 24 hr respectively. On the other hand, 20 μM these two coffee diterpenes brought about moderate up-regulation of the ATRA-induced O₂^--generating ability. Quantitative RT-PCR and immunoblotting revealed that these two coffee diterpenes significantly up-regulate the ATRA-induced O₂^--generating ability via enhancing gene expression levels of gp91-phox, which is an essential factor for the O₂^--generating ability of leukocytes. These findings demonstrated that cafestol and kahweol show not only the ATRA-enhanced cytotoxicity but also the promoting effects on the ATRA-induced O₂^--generating ability via up-regulation of gp91-phox gene expression.

Recent studies demonstrated that upregulation of proinflammatory cytokines were associated with neurotoxicity of acrylamide, which is used widely in industries and generated in food cooked at high temperature. The interleukin-1 (IL-1) is one of cytokines that play an important role in immune response. Anakinra is an IL-1 receptor antagonist used as anti-inflammatory medicine against inflammatory diseases such as juvenile idiopathic arthritis. In this study, ten-week old wild type male mice were allocated into 6 groups. Group 1 to 3 daily received subcutaneous injection with vehicle and oral exposure to ACR in drinking water at 0, 150 or 300 ppm for 28 days, and group 4-6 daily received injection with Anakinra and oral exposure to ACR in drinking water at 0, 150 or 300 ppm for 28 days. The landing foot spread (LFS) test was carried out to assess the motor function, and immunohistochemistry was carried out for quantification of noradrenergic axons and microglia activation. The results of LFS did not show significant effect of Anakinra treatment on ACR-induced increase in landing foot spread in mice. The body weight was dose-dependently decreased by ACR exposure only in the groups treated with Anakinra. The IHC staining for microglia and noradrenergic axon density does not show any significant effect of treatment with Anakinra or exposure to ACR. The study demonstrated that daily treatment with Anakinra at 25 mg/kg body weight does not ameliorate ACR-induced neurotoxicity in mice, while potentiates ACR-induced loss of body weight.

Monascus Color Y-001, a natural food pigment produced from Monascus purpureus Y-001 fermentation, was administered orally by gavage to 4 Beagle dogs/sex/group for 90 days at doses of 0 (vehicle: 0.1% Tween 80, 10 mL/kg bw), 1, 5 and 25 mg/kg bw/day. In the clinical observations, vomiting/vomitus (dark red watery substance or undigested feed) was observed for males and females in the 25, but not 5 or 1 mg/kg bw/day group at a relatively high frequency. In addition, loose stools were observed for males in this group on a single occasion. These signs were considered to be effects of the test article on the digestive tract. No treatment-related effects were noted in the detailed observations for symptoms, body weights, food consumptions, ophthalmology, hematology, clinical chemistry, urinalysis, gross pathology, organ weights or histopathology. Thus, the no-observed-adverse-effect level (NOAEL) was judged to be 5 mg/kg bw/day in both sexes of the dogs.

Subretinal injection is widely used in gene therapy research on age-related macular degeneration and retinitis pigmentosa. We investigated a method of injection and the maximum feasible volume for subretinal injections in rats and monkeys to support future studies for evaluation of new therapies in these animals. Physiological saline was injected subretinally into the eyes of male adult rats (n = 3 eyes/group) and cynomolgus monkeys (n = 3 eyes/group). The eyes were then examined by ophthalmoscopy (slit lamp and ocular fundus examinations), optical coherence tomography (OCT), intraocular pressure (IOP) measurements, or electroretinography (ERG). Rats received physiological saline at 1, 2, 5 µL/eye (2 sec/eye, bolus) and 5 µL/eye (5 µL/min). Monkeys received physiological saline at 50, 100, 150, 200, and 250 µL/eye (10 µL/sec). In all rats and cynomolgus monkeys, successful injection was visually confirmed by bleb formation using OCT at the appropriate sites immediately after injection. IOP increased more than 2-fold after injection of 5 µL/eye (5 µL/2 sec) compared with before injection but remained virtually unchanged at other volumes. Monkeys that received 200 or 250 µL/eye showed a marked increase in IOP immediately after injection, with functional abnormality observed in ERG. In conclusion, bleb formation at the appropriate sites was confirmed by OCT, demonstrating successful subretinal injections to rat and monkey eyes. When physiological saline solution is subretinally injected, the maximum feasible volume is considered to be 5 µL/eye (5 µL/min) in rats and 150 µL/eye (10 µL/sec) in cynomolgus monkeys, based on IOP and ERG results.

The head twitch response (HTR), a rapid reciprocal head movement, is a reliable behavioral indicator in rodents following the administration of hallucinogens. It is considered a model for predicting psychedelic activity in humans. A recently developed magnetometer system, consisting of a head-mounted magnet and a magnetometer coil, offers a promising alternative to laborious manual counting of HTR in animal experiments; however, it requires relatively complex analytical processes. In this study, our aim was to explore a simple and practical application of this system in ICR mice. Mice implanted with magnets were administered lysergic acid diethylamide (LSD) or (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), and their HTRs were recorded using the magnetometer system. The outputted wave signals were subjected to a 2-step screening: cutoffs for frequency band and amplitude, and manual sorting of the resulting waves. Validation through video scoring revealed a high detection accuracy of the magnetometer system, reaching nearly 99%. Next, we conducted detailed dose-response analyses for the two psychedelics at a dose range of 0.01–3 mg/kg (LSD) or 0.1–25 mg/kg (DOI). The dose-response curves displayed typical inverted U-shaped patterns, and the ED₅₀ value was calculated as 0.086 mg/kg for LSD and 1.42 mg/kg for DOI. Lastly, we explored a simpler screening method using a relatively strict set of filtering criteria instead of the 2-step screening process. A series of validation tests demonstrated that this method can detect HTR with a mean error rate of 11.20%. Our findings provide an option for the application of the magnetometer system and offer fundamental information about experimental conditions suitable for conducting HTR tests in ICR mice.

Serum separation gels are problematic in therapeutic drug monitoring because they adsorb some of the target drugs; however, the adsorptive properties of drugs that cause clinical intoxication remain unelucidated. Drug adsorption to separators results in a decrease in the observed blood levels, which may lead to uncertain assessments of clinical toxicology. Therefore, this study aimed to clarify the effects of four brands of blood collection tubes with serum separation gels that are used in Japan on the blood concentrations of central nervous system-acting drugs. Amitriptyline-, amoxapine-, mirtazapine-, chlorpromazine-, and flunitrazepam-spiked plasma at respective intoxicated concentrations were incubated in blood collecting tubes with serum separators, namely Vacutainer, Neotube, Insepack, and Venoject, at 4°C or 25°C for up to 72 or 168 hr and compared with control tubes without a serum separator. The amitriptyline, chlorpromazine, and flunitrazepam concentrations significantly decreased, even in control tubes. All the tubes containing serum separators significantly reduced the observed drug concentration when incubated at 25°C, which was estimated using a power function. Except amoxapine, rest all drugs when incubated at 4°C showed a decrease in concentration, albeit to a lesser degree than at 25°C. The blood collection tube with the greatest decrease in concentration was the Vacutainer. In conclusion, although the possibility of drug degradation in plasma must be considered, control tubes are strongly recommended for clinical toxicology, in addition to therapeutic drug monitoring, because drug adsorption is less likely to occur in these tubes.

In toxicity studies, it is important to select a vehicle that does not affect the toxicity assessment of the test substance. To obtain toxicity information on corn oil as a Vehicle for subcutaneous toxicity studies of non-aqueous test substances, Crl:CD (SD) rats (6 animals/sex/group) were repeatedly dosed with corn oil at 0 (negative control; saline), 1, 2 and 5 mL/kg/day for 4 weeks. High body weight due to retention of the administered substance were found in corn oil groups in both sexes at 2 mL/kg/day or higher. Some relative organ weights at 2 mL/kg/day or higher were decreased due to high body weight or body weight gain. Subcutaneous retention of the administered substance found in all corn oil-treated groups at necropsy was observed on histopathological analysis as accumulation of the administered substance and granulation tissue. Accumulation of the administered substance in inguinal skin, the axillary lymph node, and alveolus of the lung and bronchus was observed. Multiple white nodules in the abdominal cavity and liver observed in a female at 5 mL/kg/day at necropsy were considered to be related to lipogranuloma at the peritoneum and hepatic capsule. In hematology, RBC was lower in females at the 5 mL/kg/day than in the control group.. In conclusion, since the effects of corn oil administration were found in groups at all volumes, it is necessary to take into account that corn oil induces these changes when used as a vehicle of a test substance in toxicity studies.

Intravitreal (IVT) injections are the current method for retinal drug delivery and have been widely used in humans, although it is well known that IVT injections often cause an increase in intraocular pressure (IOP) that closely relates to the injection volume. Because of the anatomical and physiological similarities of the monkey and human eye, cynomolgus monkeys are often used for preclinical studies on new treatments using large molecules or gene therapy requiring IVT application. However, there is only limited information on the maximal dosing volume for IVT injections in cynomolgus monkeys. To determine an appropriate maximal dosing volume for IVT injections, comprehensive ocular examinations were conducted in IVT-injected eyes of cynomolgus monkeys. Up to a dosing volume of 150 µL/eye by IVT injection, there were no IVT injection-related ocular findings in pupillary light reflex gross observations, slit lamp biomicroscopic and indirect ophthalmoscopic and fundus autofluorescent examinations, intraocular pressure, optical coherence tomography of the anterior and posterior segments of the eye, axial length measurement, electroretinogram waveforms, or histopathological examinations of treated eyes. However, leakage of the dosing solution from an eye was observed at 150 µL/eye, indicating a technical limitation for the maximal applicable volume. In addition, recovery from IOP elevation immediately after injection of 150 µL/eye was slower than after injections of 50 or 100 µL/eye. Therefore, we recommend a maximal volume of 100 µL/eye for IVT injections in cynomolgus monkeys.

The erroneous assumption that herbal products is generally safe for consumption, is a major factor leading to the increased of herb-induced liver injury (HILI). Even though Laurus nobilis or laurel is a commonly used spice, the safety aspect for its consumption is under-studied. To bridge this gap of knowledge, the mutagenicity, acute toxicity, and subacute toxicity of LAURESH^®, which is a standardized laurel leaf extract were evaluated. Mutagenicity study using two S. typhimurium strains, TA100 and TA98 indicated that LAURESH^® does not cause base substitution and frameshift mutation, thus suggesting that LAURESH^® is non-mutagenic. While acute oral toxicity on mice established the LD₅₀ at no less than 2,000 mg/kg of body weight, and a 28-day subacute toxicity test on rat revealed the NOAEL to be 1,000 mg/kg/day. Furthermore, blood chemistry, urinalysis, necropsy, and histopathological data from subacute toxicity study on rats does not show adverse event that could be attributed to LAURESH^®, thus indicating that LAURESH^® is unlikely to cause HILI. Taken together, the findings from this study and previous clinical study on LAURESH^®, in combination with the historic use of laurel and previous toxicity studies conducted on laurel leaves extract, strongly suggest that LAURESH^® is safe for human consumption.

Some types of multiwalled carbon nanotube (MWCNT) are similar to asbestos in length and diameter. When these fibers are introduced into the respiratory tract, MWCNTs can induce pulmonary lesions including inflammation, fibrosis, and hyperplasia. By using an intrapulmonary spraying method, we demonstrate that MWCNT causes lung cancer in a 2-year experimental protocol (Suzui et al., 2016). In samples of the 5 histologically diagnosed archival lung cancer tissues and 4 control normal lung tissues, we examined the mRNA expression level of specific cytokines such as CCL4, IL6, and CXCL2. These cytokines were chosen for the analysis since their mRNA expression levels are upregulated in MWCNT-treating macrophages (Sultana et al., 2023). The level of expression of CCL4 markedly decreased in adenocarcinoma compared to that of the control normal lung tissue. In several adenocarcinoma samples, the level of expression of IL6/CXCL2 was higher than that of the control normal lung tissue. In the granuloma sample, the level of IL6 was higher than that of the control normal lung tissue and the level of CCL4/CXCL2 was lower than that of the control normal lung tissue. Taken together, histology specific expression profile of these cytokines may provide additional insights into lung tumorigenesis induced by MWCNT.

Cadmium (Cd) is an environmental pollutant with toxic effects in various tissues, including the kidney. In our previous study, Cd changed the expression of baculoviral inhibitor of apoptosis protein repeat-containing (BIRC) family genes in human proximal tubular cells. BIRC family genes inhibit apoptosis, and inducing the apoptosis pathway is an approach for anticancer therapeutics. In this study, the effect of Cd on expression of BIRC family genes was examined in HeLa human cervical cancer cells. Cd treatment affected cell viability, increased BIRC5 and BIRC6 mRNA levels, and decreased the BIRC8 mRNA level in HeLa cells. These results indicate that a low dose of Cd changes the expression of several genes in the BIRC family in HeLa cells. Changes in the mRNA levels of BIRC family genes by exposure to Cd may affect cancer chemotherapeutic agents targeting the apoptosis pathway.

The blood-brain barrier restricts the administration of drugs for neurological diseases. K16ApoE is an effective drug delivery carrier to deliver drugs across the blood-brain barrier, but it contains acute and high toxicity. The toxicity mechanism of K16ApoE must be revealed for clinical uses. Previous studies hypothesized that the toxicity mechanism was acetylcholinesterase inhibition in the brain. However, these studies used improper buffers in the AChE assay, further leading to anomalous results. Meanwhile, previous studies have not investigated the effects of K16ApoE on all the AChE-containing tissues and organs throughout the body. The previous dose design was also too narrow. Herein, we designed a more comprehensive and rational dose interval for K16ApoE, observed and recorded the mouse responses after receiving K16ApoE, and collected the brain, diaphragm, and serum to investigate the systemic K16ApoE effects on AChE. We also incubated purified AChE with K16ApoE in vitro. It could reveal the direct effect of K16ApoE on AChE without the influence of absorption and metabolism in vivo. The in vitro results demonstrated that K16ApoE inhibits rhAChE activity as the dose increases. However, the in vivo results demonstrated that K16ApoE does not affect tissue AChE activity in female mice. Therefore, we confirmed the AChE inhibitory effect of K16ApoE, but contrary to our hypothesis, AChE inhibition is not the toxicological mechanism of K16ApoE. We also recorded toxicological responses after the mice received K16ApoE, which would promote further toxicological investigation on K16ApoE.

Alternatives to animal testing are being used to assess the safety of raw ingredients during the process of developing cosmetics. However, since cosmetic products are composed of a variety of raw ingredients, the safety of the product itself should also be tested. In this study we attempted to evaluate the skin irritation potential of skin lotions by modifying existing alternative test methods. To evaluate the skin irritation potential of commercial skin lotions in the form of an objective indicator, we calculated their Irritation Score (IS) based on the results of searches using keywords related to skin irritation in review statements posted on Japanese cosmetics and beauty websites. We then modified the reconstructed human epidermis (RhE) test method to evaluate irritation by commercial skin lotions. The results showed that exposure to skin lotions with higher ISs tended to result in lower cell viability, and that exposure to many of the lotions with lower ISs resulted in higher cell viability. Next, we tried using an in vitro short-term exposure (STE) test method to assess the skin irritation potential of skin lotions. By changing the test-substance exposure concentrations and exposure times in the STE test method, we were able to obtain results that correlated with those obtained by the modified RhE test method. In conclusion, both alternative methods were helpful for assessing the possibility of developing skin irritation of skin lotions. They may also be useful for screening formulations being developed and as means of evaluation before proceeding to human patch tests.

For the non-clinical safety evaluation of pharmaceuticals for new drug applications (NDA), various toxicity studies must be conducted at each stage, from clinical trials to NDA. For topically applied drugs, the level of exposure at the administration site is high because the drug is administered directly to the administration site. However, because systemic exposure to ophthalmic drugs is generally lower than that of systemic drugs, systemic effects may not be adequately assessed. The bone marrow and liver are generally evaluated after systemic administration in in vivo genotoxicity tests, and local genotoxicity studies are conducted on a case-by-case basis. Therefore, we surveyed packages of genotoxicity and carcinogenicity tests for ophthalmic drugs approved in Japan from 2004 to 2021 to assist in the decision of test packages for the development of ophthalmic drugs. There were no major differences in genotoxicity test packages compared to systemic drugs; however, an unscheduled DNA synthesis test using the cornea after ocular instillation was conducted in some products as a test specific to ophthalmic drugs. In the development of ophthalmic drugs, if a positive result is found in an in vitro genotoxicity test, the safety margin between the positive concentration and the clinically applicable concentration (eye drop concentration) is required for safety assessment. If the safety margin cannot be ensured, additional tests may support safety assessment.

1,2-Dichloroethane, a priority assessment chemical substance under the Japan Chemical Substances Control Law (CSCL), required a detailed human health hazard assessment under Assessment II. We evaluated its general, reproductive, and developmental toxicities, genotoxicity, and carcinogenicity, based on the hazard information provided by domestic and international risk assessment organizations, and the hazard assessment values (HAVs) for oral and inhalation exposure were proposed. For oral exposure, a 78-week gavage carcinogenicity study (US NCI, 1978) with incidence data of hemangiosarcoma in male rats was selected as a significant toxicological endpoint and the lower confidence limit of benchmark dose (BMD) at 10% benchmark response (BMDL₁₀) of 9.3 mg/kg/day was obtained as a point of departure (POD). A slope factor of 1.07 × 10⁻² (mg/kg/day)⁻¹ from which a carcinogenic 10⁻⁵ risk of 0.93 µg/kg/day was derived as an oral HAV. For inhalation exposure, a 104-week inhalation exposure carcinogenicity study (Nagano et al., 2006) with a BMDL₁₀ of 11.5 ppm based on the incidence data of mammary gland tumors (adenocarcinoma + adenoma + fibroadenoma, combined) in female rats was obtained, and the human equivalent BMDL₁₀ of 15.7 mg/m³ was calculated. Therefore, a unit risk of 6.40 × 10⁻⁶ (µg/m³)⁻¹ from which a carcinogenic 10⁻⁵ risk of 1.6 µg/m³ (0.00039 ppm) was derived as an inhalation HAV.

Editor’s Announcement

Quantitative morphometric analysis of vimentin filaments in Sertoli cells of rats after in utero DBP exposure

Eisuke Kume, Yuya Okayama, Mituru Sugiyama, Hiroyuki Takahashi, Tomoko Muto, Michael F. Wempe, Hiroshi Ikegami, Shin Wakui

(Fundamental Toxicological Sciences, 4, 85-93, 2017)

I have retracted the above paper as Editor-in-Chief of Fundamental Toxicological Sciences since I have serious concerns about it, primarily due to inappropriate authorship on a non-negligible scale.

Since the possibility of inappropriate authorship in this paper was raised, I contacted the co-authors to confirm this point. I found out that several of them considered their listing as co-authors to be inappropriate. In addition, more than half of the co-authors agreed to the retraction of this paper.

These facts raise concerns about the paper. From the standpoint of maintaining the integrity of the research community, I felt that such a paper should be retracted at once.

Accordingly, I sent a summary of my concerns about this paper to the corresponding author, Dr. Shin Wakui. I also had an online interview with him to discuss this matter. I told Dr. Wakui that inappropriate authorship on a non-negligible scale is a serious problem that raises concerns about the paper.

I prepared a draft of this Editor’s Announcement and sent it to Dr. Wakui for review prior to revision and release. Although he did not agree to the retraction, I have decided to take this action from the standpoint of maintaining the integrity of the research community.

I coordinated my response to this issue with Dr. Toshiyuki Kaji, Editor-in-Chief of The Journal of Toxicological Sciences, a sister journal of Fundamental Toxicological Sciences.

Akira Naganuma, Ph.D.
Editor-in-Chief
Fundamental Toxicological Sciences

Aside from the COVID-19 pandemic, the obesity and diabetes pandemics have threatened global health. Patients with diabetes are more likely to experience serious complications from COVID-19; thus, preventing obesity-associated diabetes is of paramount important. Furthermore, the development of a method to prevent diabetes and elucidation of its pathology is a currently urgent issue. We previously reported that thiamine plays a key role in suppressing abnormal glycolipid metabolism in Otsuka Long-Evans Tokushima fatty (OLETF) rats, an animal model of obesity-associated diabetes. However, whether thiamine affects only OLETF rats or other animal models including a type 2 diabetes model with a different pathology requires elucidation. In this study, leptin-receptor deficiency mice were used as a model of type 2 diabetes with a different pathology to evaluate the efficacy of thiamine. The mice had free access to water containing 0.2% thiamine for 9 weeks, and the results showed that food and water consumption decreased in db/db-homo mice. Urine output, body weight gains and blood glucose levels decreased in mice that received thiamine. There were 5 mice and 1 mouse with a fasting glucose level of ≥ 300 mg/dL in the db/db-homo control group (n = 10) and db/db-homo thiamine group (n = 10), respectively, suggesting that thiamine intake may suppress an increase in blood glucose levels. The results of the present study suggest that demand for thiamine may exceed the normal range in in vivo mouse models of diabetes and continuous thiamine intake affects diabetes onset and progression.

2-Butylbenzo[d]isothiazol-3(2H)-one (BBIT, CAS No. 4299-07-4) is widely used as an industrial antiseptic and antifungal agent. To investigate its toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral toxicological study of BBIT was conducted in Crl:CD (SD) rats at doses of 0 (vehicle control, corn oil), 30, 90, or 270 mg/kg/day. There was no mortality or abnormal clinical signs related to treatment in any group. Slightly decreased body weight and food consumption were observed in the 270 mg/kg group in females. Increased urine volume and kidney weight, increased liver weight, and thickening of the forestomach mucosa in autopsy were observed in both sexes in the 270 mg/kg group. Histopathological examination revealed that hyperplasia of the squamous epithelium of the forestomach with parakeratosis and/or hyperkeratosis was observed in both sexes in all the BBIT-treated groups. Moreover, centrilobular hypertrophy of hepatocytes was observed in both sexes of the 270 mg/kg group. Similarly, increased depositions of eosinophilic bodies and/or hyaline droplets in the proximal tubules of the kidney were observed among the male in the 270 mg/kg group. Based on the forestomach changes, NOAEL was judged to be less than 30 mg/kg/day in both sexes under this study’s conditions.

2-(2H-benzotriazol-2-yl)-6-dodecyl-4-methylphenol (BTMLP, CAS No. 125304-04-3) is widely used as a liquid ultraviolet absorber that prevents deterioration of synthetic resins and so on. To investigate its toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral toxicological study of BTMLP was conducted in Crl:CD (SD) rats at doses of 0 (vehicle control, corn oil), 100, 300, and 1000 mg/kg/day. There was no observed mortality or abnormal clinical signs related to the treatment of any group. Body weight and food consumption were not affected by BTMLP treatment. In males, significant prolongations of prothrombin time and activated partial thrombin time were observed in the BTMLP-treated groups. Histopathological examination revealed a slight increase of the eosinophilic bodies and hyaline droplets in the renal cortical tubules in the 1000 mg/kg group in males. As mentioned above, the toxic effect of the BTMLP was noted in the blood coagulation system and kidneys only in males. Based on these findings, the NOAEL was judged to be less than 100 mg/kg/day in males and 1000 mg/kg/day in females under this study’s condition.

To investigate the effects of Chlorella alga on gut microbiota dysbiosis in type-2 diabetes (T2D). The stress perception of patients was also investigated. Chlorella (3 g/day) was administered to patients with T2D (n = 10) for a period of 30 days. Gut microbiota composition was analysed by 16S rDNA gene sequencing, and stress perception was evaluated using the perceived stress scale (PSS). A total of 13 phyla, 89 families, and 185 genera were identified from all faecal samples of patients with T2D, and Firmicutes and Bacteroidetes were the most dominant phyla among all samples. Chlorella decreased Bacteroidetes and increased Firmicutes. The proportions of the Akkermansia, Coprococcus, Dorea, Lachnospira, Phascolarctobacterium, and Ruminococcus generas increased, whereas the proportion of Paraprevotella, Prevotella, Klebsiella, and Sutterella decreased in the faeces of patients with T2D after Chlorella intake. Chlorella induced a significant reduction in perceived stress in patients with T2D, and better PSS scores negatively correlated with an increase in Akkermansia, Coprococcus, Dorea, Lachnospira, Phascolarctobacterium, and Ruminococcus and positively correlated with a decrease in Paraprevotella, Prevotella, Klebsiella, and Sutterella. Altogether, these results show the ability of Chlorella to positively modulate gut dysbiosis, leading to reduced stress perception in patients with T2D. Our findings contribute to the globally increasing search for new preventive and therapeutic strategies aimed at restoring the balance of the intestinal ecosystem.

The long-term effects of Chlorella doses on the inflammatory status and quality of life (QoL) of individuals with type-2 diabetes (T2D), and prediabetes (pre-T2D), and of nondiabetic controls were investigated. Chlorella was administered for 12 months; 1.6 g/day for the first six months and 3 g/day for the following six months. The inflammatory profile was studied by quantification of cytokines, adipokines and incretins. QoL was evaluated using the Short Form-36 health survey questionnaire (SF-36). Evaluations were performed at baseline, 6 (T6) and 12 (T12) months after initiating Chlorella intake. At baseline, QoL was more deeply impacted in T2D, a similar proinflammatory profile was observed in T2D and pre-T2D. In both, at T6 and T12, Chlorella modulated the altered levels of adipocytokines and incretins towards healthy values, and significantly improved QoL. Moderate correlations between the modulation by the alga and enhancement in QoL were observed only in the T2D group. In the nondiabetic control group, Chlorella improved QoL vitality and mental health scores. No differences were found between the two doses. Our results illustrate Chlorella adaptogen activity on inflammatory pathways and suggest its promising use as a complementary alternative in treating diseases related to insulin resistance in a wide range of chronic low-grade systemic inflammation-related diseases. Moreover, Chlorella increased QoL in all groups, the ultimate goal of all healthy interventions. Altogether, our findings suggest that one core mechanism involved in the homeostatic response produced by Chlorella is related to its rich content of carotenoids, operating mainly through inhibition of the NF-κB signalling pathway.

Multiwalled carbon nanotubes (MWCNT) are fiber-shaped nanomaterials that have a potential risk for cancer due to properties that are similar to asbestos. One type of nanotube called MWCNT-7 was categorized in Group 2B as possibly carcinogenic to humans by the International Agency for Research on Cancer. MWCNT-N, which is similar to MWCNT-7, is carcinogenic to the lung and pleura when administered to rats via the respiratory tract using intra-tracheal intra‐pulmonary spraying. Macrophages have an important role in the MWCNT induced pulmonary carcinogenicity. In this study, rat primary alveolar macrophages were employed to examine possible mechanism of carcinogenic effects of the MWCNT-N. MWCNT-N was fractionated into flow-through and retained fraction by passing it through a sieve with a pore size of 25 μm. Microarray analysis showed up-regulation of various cytokines in macrophages exposed to MWCNT-N. MWCNT-N strongly induced the expression of cytokines such as interleukin-6 (IL-6) in macrophages. In contrast, few cytokines were prominently down-regulated in macrophages treated with MWCNT-N. The sieve fractions did not have a significant effect on mRNA expression of cytokines in macrophages. These results provide useful information for understanding MWCNT-induced carcinogenicity via cytokine expression by macrophages.

DNA damage may be induced by both intrinsic and extrinsic factors, the latter involving chemical exposure at workplaces. Upon DNA damage induction, checkpoint kinases such as ATM and ATR, phosphorylate serine 139 of the histone H2AX generating γH2AX, to initiate the damage response pathway. This allows antibodies that act against post-translational modifications, such as γH2AX, to be used for detecting genotoxicity. The aromatic amine 4,4'-methylenebis(2-chloroaniline) (MOCA), which is utilized in industry to produce polyurethane resins, exhibits genotoxicity and induces cancers in animals, including rodents and humans. DNA damage, due to MOCA-induced toxicity during its metabolism, is believed to be first step towards carcinogenesis. However, we failed to detect γH2AX induction by MOCA in cellular systems via western blotting, even when electrophoresis-based methods clearly indicated that physical DNA damage had been induced. In the present study, we utilized RNA sequencing of MOCA-treated cells and controls to elucidate factors underlying the discrepancies observed between these two analytical systems. Our results indicated that H2AX mRNA expression was significantly downregulated; this finding partially clarified the inefficient induction of γH2AX. Although downregulation of γH2AX in the presence of MOCA witnessed in this study was quite unexpected, we believe that this finding prompts researchers to be cautious when screening for genotoxicity using γH2AX only, due to the possibility of genotoxicity being overlooked in some cases. Thus, it appears that a combined approach may be more suitable for detecting genotoxicity.

Methylphenidate (MPH) is used as a first-line treatment for attention-deficit/hyperactivity disorder (ADHD). Because the onset of ADHD appears in early childhood and the incidence number is increasing, more patients could become adults with long-term use of MPH. In addition, few men would discontinue the medication during a fertile period. Recently, environmental factors such as diet and drug abuse have been reported to produce changes in the sperm epigenome and affect the health of the next generation. However, the effects of long-term administration of a psychostimulant such as MPH on the next generation is unknown. In this study, we examined the effects of paternal administration of MPH on the growth, behavior, and gene expression in offspring using a mouse model. Sires were subcutaneously administered MPH for 21 days and mated with naive dams. Upon reaching 6–7 weeks of age, offspring were subjected to spontaneous locomotor, elevated plus-maze, and passive avoidance tests. Additionally, RNA-seq and RT-qPCR were performed on the striatum. Paternal MPH exposure induced increased atomoxetine-sensitive impulsivity and decreased long-term memory function in the offspring. Enrichment analysis following RNA-seq revealed significant enrichment of terms involved in the nervous system. Gene expression levels of Snap25, Syt1, Drd2, Maoa, and Comt, which are associated with ADHD pathology, are altered in the striatum. These results suggest that continuous administration of MPH to male mice induces ADHD-like behavior and changes in the expression of genes involved in the nervous system in the brain of the next generation.

TRPM8, non-selective cation channel of the transient receptor potential (TRP) superfamily, required for the transduction of moderate cold temperatures, regulates proliferation of epidermal cells in cyclin-dependent kinase inhibitor p21/Cip1-dependent manner. Given that downregulation of TRPM8 decreases p21/Cip1 level, increasing risk for carcinogenesis, and other TRP family is regulated by nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma, we examined whether TRPM8 expression was regulated by PPAR gamma. Knockdown assay and inhibition of PPAR gamma revealed that PPAR gamma negatively regulates the expression of TRPM8 in normal epidermal cells but positively regulates that in squamous carcinoma cells. Later restoration of decreased TRPM8 level in PPAR gamma antagonist-treated squamous carcinoma cells was attributed to feed-back loop regulation between TRPM8 and PPAR gamma using TRPM8 knockdown assay. Consisting with this finding, p21/Cip1 decrease by TRPM8 blocker, N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin- 2-yl) tetrahydropyrazine-1 (2H)-carbox-amide (BCTC), was restored by additional BCTC treatment in squamous carcinoma cells.