Assessment of commercial polymers with and without reactive groups using amino acid derivative reactivity assay based on both molar concentration approach and gravimetric approach.

The amino acid derivative reactivity assay (ADRA), an alternative method for testing skin sensitization, has been established based on the molar concentration approach. However, the additional development of gravimetric concentration and fluorescence detection methods has expanded its range of application to mixtures, which cannot be evaluated using the conventional testing method, the direct peptide reactivity assay (DPRA). Although polymers are generally treated as mixtures, there have been no reports of actual polymer evaluations using alternative methods owing to their insolubility. Therefore, in this study, we evaluated skin sensitization potential of polymers, which is difficult to predict, using ADRA. As polymers have molecular weights ranging from several thousand to more than several tens of thousand Daltons, they are unlikely to cause skin sensitization due to their extremely low penetration into the skin, according to the 500-Da rule. However, if highly reactive functional groups remain at the ends or side chains of polymers, relatively low-molecular-weight polymer components may penetrate the skin to cause sensitization. Polymers can be roughly classified into three major types based on the features of their constituent monomers; we investigated the sensitization capacity of each type of polymer. Polymers with alert sensitization structures at their ends were classified as skin sensitizers, whereas those with no residual reactive groups were classified as nonsensitizers. Although polymers with a glycidyl group need to be evaluated carefully, we concluded that ADRA (0.5 mg/ml) is generally sufficient for polymer hazard assessment.

Hypothyroidism due to nephrotic syndrome: a notable clinical entity.

Nephrotic syndrome (NS) is characterized by massive urinary protein leakage and associated hypoproteinemia due to increased protein permeability caused by impaired renal glomerular connections. Although there have been several sporadic reports regarding the relationship between NS and thyroid dysfunction, a consensus has yet to be reached. The mechanism of hypothyroidism in NS is attributed to the loss of protein-bound thyroid hormones, such as thyroxine-binding globulin, transthyretin, and albumin, into the urine. Herein, we report four adults with hypothyroidism that developed or worsened due to the onset of NS. The patients' underlying thyroid status was post-total thyroidectomy with supplemental levothyroxine (L-T4) in two patients, hypothyroidism with supplemental L-T4 due to Hashimoto's disease in one patient, and Hashimoto's disease with normal thyroid function in one patient. Our results suggest that the presence of a reduced thyroid reserve may predispose patients to hypothyroidism in NS. We conclude that NS may cause or exacerbate hypothyroidism. In such cases, an NS assessment, including a urine test, is required.

The magnitude of increased Levothyroxine dose during pregnancy in patients on thyroid-stimulating hormone (TSH) suppression treatment after total thyroidectomy for papillary carcinoma.

The dose of L-T4 replacement for hypothyroidism often needs to be increased after pregnancy. In our institution, patients are instructed to double the dose 2 days a week after pregnancy. However, there is scarce evidence supporting the need for a dose increase after pregnancy in patients with preconception thyroid-stimulating hormone (TSH) suppression (TSH <0.3 µIU/mL). This study aimed to determine the need for a dose increase in L-T4 among women with a TSH-suppressive dose of L-T4 before pregnancy. In this retrospective observational study, between January 2008 and December 2018, we analyzed 166 pregnancies in 134 patients on TSH suppression treatment after total thyroidectomy for papillary carcinoma. Thyroid function tests were performed before and in the first trimester of pregnancy. The dose was adjusted and maintained during the first trimester of pregnancy in 76 pregnancies (group A) and 90 pregnancies (group B), respectively. The median serum TSH level was significantly lower in group A than that in group B (0.014 µIU/mL (IQR, 0.005-0.071) vs. 0.155 µIU/mL (IQR, 0.021-0.657), p < 0.001). TSH suppression could not be maintained after pregnancy in 15.8% and 38.9% of the pregnancies in groups A and B, respectively. Increasing the post-pregnancy dose by an average of 27.4% resulted in maintenance of TSH suppression after pregnancy in 84.2% of pregnancies. In conclusion, this study suggests that increasing the L-T4 dose after pregnancy may be appropriate in postoperative thyroid cancer patients whose serum TSH levels should be suppressed.

Applicability of amino acid derivative reactivity assay (4 mM) for the prediction of skin sensitization by combining multiple alternative methods to evaluate key events.

The amino acid derivative reactivity assay (ADRA) is an alternative method for evaluating key event 1 (KE-1) in the skin sensitization mechanism included in OECD TG442C (OECD, 2021). Recently, we found that ADRA with a 4-mM test chemical solution had a higher accuracy than the original ADRA (1 mM). However, ADRA (4 mM) has yet to be evaluated using integrated approaches to testing and assessment (IATA), a combination of alternative methods for evaluating KE. In this study, the sensitization potency of three defined approaches (DAs) using ADRA (4 mM) as KE-1 was predicted and compared with those of two additional ADRAs or direct peptide reactivity assay (DPRA): (i) "2 out of 3" approach, (ii) "3 out of 3" approach, and (iii) integrated testing strategy (ITS). In the hazard identification of chemical sensitizers, the accuracy of human data and local lymph node assay (LLNA) remained almost unchanged among the three approaches evaluated. Potency classifications for sensitization were predicted with the LLNA and human data sets using ITS. The potency classifications for the sensitization potency prediction accuracy of LLNA data using any alternative method were almost unchanged, at approximately 70%, and those with ITS were not significantly different. When ITS was performed using DPRA, the prediction accuracy was approximately 73% for human data, which was similar to that of the LLNA data; however, the accuracy tended to increase for all ADRA methods. In particular, when ITS was performed using ADRA (4 mM), the prediction accuracy was approximately 78%, which proved to be a practical level.

Within- and between-laboratory reproducibility and predictive capacity of amino acid derivative reactivity assay (ADRA) using a 0.5 mg/mL test chemical solution: Results of the study for reproducibility confirmation implemented in five participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative assay for skin sensitization listed in OECD test guideline 442C. ADRA evaluates the reactivity of sensitizers to proteins, which is key event 1 in the skin sensitization adverse outcome pathway. Although the current key event 1 evaluation method is a simple assay that evaluates nucleophile and test chemical reactivity, mixtures of unknown molecular weights cannot be evaluated because a constant molar ratio between the nucleophile and test chemical is necessary. In addition, because the nucleophile is quantified by HPLC, the frequency of co-eluting the test chemical and nucleophile increases when measuring multi-component mixtures. To solve these issues, test conditions have been developed using a 0.5 mg/mL test chemical solution and fluorescence-based detection. Since the practicality of these methods has not been substantiated, a validation test to confirm reproducibility was conducted in this study. The 10 proficiency substances listed in the ADRA guidelines were tested three times at five different laboratories. The results of both within- and between-laboratory reproducibility were 100%, and the results of ultraviolet- and fluorescence-based measurements were also consistent. In addition to the proficiency substances, a new positive control, squaric acid diethyl ester, was tested three times at the five laboratories. The results showed high reproducibility with N-(2-(1-naphthyl)acetyl)-l-cysteine depletion of 37%-52% and α-N-(2-(1-naphthyl)acetyl)-l-lysine depletion of 99%-100%. Thus, high reproducibility was confirmed in both evaluations of the 0.5 mg/mL test chemical and the fluorescence-based measurements, validating the practicability of these methods.

The within- and between-laboratories reproducibility and predictive capacity of Amino acid Derivative Reactivity Assay using 4 mM test chemical solution: Results of ring study implemented at five participating laboratories.

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

Cause Clarification of Cysteine Oxidation by Active Species Generated during the Oxidation Process of Cinnamaldehyde and Impact on an In Chemico Alternative Method for Skin Sensitization Using a Nucleophilic Reagent Containing Cysteine.

Aldehydes comprise a major portion of skin sensitizers because they can react with both cysteine and lysine. Moreover, cinnamaldehyde (CA) is a typical moderate sensitizer and is often used in an alternative test method for skin sensitization. The amino acid derivative reactivity assay (ADRA) is an in chemico test method that evaluates the reactivity of cysteine derivatives (N-(2-(1-naphthyl)acetyl)-l-cysteine, NAC) and lysine derivatives with the test chemicals and uses CA as a proficiency substance. We found that NAC depletion for CA was only 10-20% when CA was used directly from the reagent bottle, although it increased to almost 100% when stored after being aliquoted from the reagent bottle. It was also found that this was due to the air oxidation of NAC itself rather than the reaction of NAC with CA, indicating that this result simply shows an increase in apparent reactivity. Aldehydes are known to produce active species, such as radicals, during air oxidation. Therefore, we investigated whether radicals were generated under storage conditions using the radical scavenger OH-TEMPO. LC/MS/MS analysis revealed that CA and OH-TEMPO complexes were produced during the air oxidation of CA. In the results of five aldehydes, similar to CA, active species were not generated as significantly as CA. Collectively, during the evaluation of the aldehydes, it can be seen that careful measures need to be taken to prevent the aldehydes from oxidizing during storage, indicating that assessment without preventing air oxidation carries an increased risk of overestimation compared with the intrinsic skin sensitization potency.

Association between serum thyroid hormone balance and thyroid volume in patients treated with levothyroxine monotherapy for hypothyroidism.

Many previous studies including ours have reported that athyreotic patients on levothyroxine (LT4) have relatively low serum free triiodothyronine (FT3) levels, whereas patients with large goitrous diseases often have high serum FT3 levels. Here we investigated Hashimoto thyroiditis (HT) patients on LT4 to study the relationship between thyroid volume (TV) and thyroid hormone status in hypothyroid patients on LT4. We retrospectively studied 408 euthyroid HT patients treated with LT4 for hypothyroidism; divided them as per TV and compared serum levels of free thyroxine (FT4) and FT3 and the FT3/FT4 ratio in each patient group with those in euthyroid matched control group. We also evaluated the association between serum FT3 level and FT3/FT4 ratio and TV among HT patients on LT4. In patients with TV <15 mL, serum FT3 levels were significantly lower than those in controls. In patients with TV 15-80 mL, serum FT3 levels were equivalent to those in controls. In patients with TV ≥80 mL, the serum FT3 levels were significantly higher than those in controls. The serum FT3 level (r = 0.35, p < 0.01) and FT3/FT4 ratio (r = 0.42, p < 0.01) showed a positive correlation with TV. TVs in HT patients on LT4 caused differences in serum thyroid hormone balance, as increasing volume increases the serum FT3 level and FT3/FT4 ratio. Serum thyroid hormone balance in HT patients with smaller thyroids was similar to that in athyreotic patients. Mild thyrotropin suppression with LT4 is needed to achieve normal FT3 levels in such patients.

Position paper from the Japan Thyroid Association task force on the management of low-risk papillary thyroid microcarcinoma (T1aN0M0) in adults.

The incidence of thyroid carcinoma has been increasing worldwide. This is interpreted as an increase in the incidental detection of papillary thyroid microcarcinomas (PTMCs). However, mortality has not changed, suggesting overdiagnosis and overtreatment. Prospective clinical trials of active surveillance for low-risk PTMC (T1aN0M0) have been conducted in two Japanese institutions since the 1990s. Based on the favorable outcomes of these trials, active surveillance has been gradually adopted worldwide. A task force on the management of PTMC in adults organized by the Japan Thyroid Association therefore conducted a systematic review and has produced the present position paper based on the scientific evidence concerning active surveillance. This paper indicates evidence for the increased incidence of PTMC, favorable surgical outcomes for low-risk PTMC, recommended criteria for diagnosis using fine needle aspiration cytology, and evaluation of lymph node metastasis (LNM), extrathyroidal extension (ETE) and distant metastasis. Active surveillance has also been reported with a low incidence of disease progression and no subsequent recurrence or adverse events on survival if conversion surgery was performed at a slightly advanced stage. Active surveillance is a safe and valid strategy for PTMC, because it might preserve physical quality of life and reduce 10-year medical costs. However, some points should be noted when performing active surveillance. Immediate surgery is needed for PTMC showing high-risk features, such as clinical LNM, ETE or distant metastasis. Active surveillance should be performed under an appropriate medical team and should be continued for life.

Amino acid derivative reactivity assay-organic solvent reaction system: A novel alternative test for skin sensitization capable of assessing highly hydrophobic substances.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing that focuses on protein binding. The ADRA is a skin sensitization test that solves problems associated with the direct peptide reactivity assay. However, when utilizing the ADRA to evaluate highly hydrophobic substances with octanol/water partition coefficients (logKow) of >6, the test substances may not dissolve in the reaction solution, which can prevent the accurate assessment of skin sensitization. Therefore, we developed the ADRA-organic solvent (ADRA-OS) reaction system, which is a novel skin sensitization test that enables the assessment of highly hydrophobic substances with a logKow of >6. We discovered that the organic solvent ratio, the triethylamine concentration, and the ethylenediaminetetraacetic acid disodium salt dihydrate concentration participate in reactions with the nucleophile N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and sensitizers that are used in ADRA and in stabilizing NAC. Thus, we determined the optimal reaction composition of the ADRA-OS according to L9 (33 ) orthogonal array experiments. Using this test, we assessed 14 types of highly hydrophobic substances. When we compared the results with ADRA, we found that ADRA-OS reaction system has high solubility for highly hydrophobic substances and that it has a high predictive capacity (sensitivity: 63%, specificity: 100%, accuracy: 79%). The implication of the results is that the novel ADRA-OS reaction system should provide a useful method for assessing the skin sensitization of highly hydrophobic substances with a logKow of >6.

Improving predictive capacity of the Amino acid Derivative Reactivity Assay test method for skin sensitization potential with an optimal molar concentration of test chemical solution.

The Amino acid Derivative Reactivity Assay (ADRA) is a convenient and effective in chemico test method for assessing covalent binding of test chemicals with protein-derived nucleophilic reagents as a means of predicting skin sensitization potential. Although the original molar-concentration approach to ADRA testing was not suitable for testing multiconstituent substances of an unknown composition, a weight-concentration approach that is suitable for such substances was developed, which also led to the realization that test chemical solutions prepared to molar concentrations higher than the original 1 mM would reduce false negative results as well as enhance predictive capacity. The present study determined an optimal molar-concentration that achieves even higher predictive capacity than the original ADRA. Eight chemicals that were false negatives when tested with 1 mM test chemical solutions were retested with test chemical solutions between 2 and 5 mM, which showed 4 mM to be the optimal molar-concentration for ADRA testing. When 82 chemicals used in the original development were retested with 4 mM test chemical solutions, false negative results were reduced by four. When an additional 85 chemicals used to evaluate the weight-concentration approach to ADRA were retested, the results essentially replicated those obtained with 0.5 mg/ml test chemical solutions and gave 10 fewer false negatives than original ADRA with 1 mM solutions. A comparison of these results for 136 chemicals showed that ADRA testing with 4 mM solutions achieved a four percentage point improvement in accuracy over original ADRA and a two percentage point improvement over DPRA testing.

Quality of Life in Patients with Low-Risk Papillary Thyroid Microcarcinoma: Active Surveillance Versus Immediate Surgery.

OBJECTIVE: This study aimed to compare the quality of life (QoL) and psychological issues of patients with papillary thyroid microcarcinoma (PMC) who were under active surveillance (AS) and those who underwent immediate surgery (OP). METHODS: This was a cross-sectional study conducted on 347 patients with low-risk PMC who were under AS (n = 298) or who underwent OP (n = 49). They were asked to complete two questionnaires (thyroid cancer-specific health-related QoL [THYCA-QoL] and the Hospital Anxiety and Depression Scale [HADS]). The results between the AS and OP groups were compared. RESULTS: The mean ages of patients in the AS and OP groups were 58.6±12.5 and 58.4±13.1 years (P =.94), respectively, and the male ratios were 34/298 (11%) and 2/49 (4.1%) (P =.14), respectively. The median follow-up periods from diagnosis in the AS and OP groups were 56.5 months (interquartile range [IQR], 32 to 88 months) and 84 months (IQR, 64 to 130 months) (P<.001), respectively. In the THYCA-QoL questionnaire, the OP group had more complaints about "voice" (P<.001), "psychological" (P =.025), "problems with scar" (P<.001), and "gained weight" (P =.047) than the AS group. Other scales of the THYCA-QoL were comparable in the two groups. In the HADS questionnaire, the AS group had significantly better anxiety (P =.020), depression (P =.027), and total scores (P =.014) than the OP group. CONCLUSION: PMC patients in the OP group had more complaints and were more anxious and depressed than the AS group. These findings suggest that AS is a reasonable alternative to surgery for patients with low-risk PMC from the point of view of QoL and psychology. ABBREVIATIONS: AS = active surveillance; CI = confidence interval; HADS = Hospital Anxiety and Depression Scale; LT4 = levothyroxine; OP = immediate surgery; PMC = papillary microcarcinoma; PTC = papillary thyroid carcinoma; QoL = quality of life; STAI = State-Trait Anxiety Inventory; THYCA-QoL = thyroid cancer-specific health-related quality of life; TSH = thyrotropin.

Development of photo-amino acid derivative reactivity assay: a novel in chemico alternative method for predicting photoallergy.

Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefore, we tried to establish a photoallergy test by utilizing an in chemico alternative sensitization method, amino acid derivative reactivity assay (ADRA). To determine the criteria for judging the photoallergy potential, photo-ADRA with or without photoirradiation was performed using 60 photoallergenic chemicals, and cysteine and lysine derivatives were detected using high-performance liquid chromatography either by absorbance or fluorescence measurement. The accuracy of prediction was 81.4% (48 of 59) and 80.0% (48 of 60) using the absorbance and fluorescence methods, respectively. However, as chemicals can breakdown into multiple chemicals during photoirradiation, the absorbance method often cannot perform accurate detection due to co-elution, whereas the fluorescence method can do this due to lack of co-elution. Moreover, all eight chemicals that were found to be negative or false-positive for photoirritation in the 3T3 neutral red uptake phototoxicity test were confirmed as positive for photoallergy using this method. Furthermore, we prepared three types of pseudo-mixtures where we added one photoallergen along with five nonphotoallergens and performed the photo-ADRA by the ultraviolet and fluorescence methods. The result of the fluorescence method was almost the same as that obtained with the use of a single photoallergen and hence the outcome was not affected by the mixture. Thus, this study not only showed a method of evaluating the photoallergy potential of a single chemical but also a mixture, making it useful as an in chemico photoallergy alternative test.

Oxidation of a cysteine-derived nucleophilic reagent by dimethyl sulfoxide in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 µm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.

The association between thyroid hormone balance and thyroid volume in patients with Hashimoto thyroiditis.

While patients with large goitrous thyroid diseases often have a relatively high serum free triiodothyronine (FT3)/free thyroxine (FT4) ratio, athyreotic patients have a relatively low FT3/FT4 ratio. Here we investigated the relationship between thyroid hormone status and thyroid volume (TV) among a large number of euthyroid Hashimoto thyroiditis (HT) patients. We retrospectively enrolled 2,603 untreated HT patients who visited the Kuma hospital from 2012 to 2016, and divided them into four groups as per the TV: normal TV (<20 mL), slight goiter (20 ≤ TV < 50 mL), moderate goiter (50 ≤ TV < 80 mL), and the large goiter group (≥80 mL). Baseline characteristics and laboratory data of each group were compared to those of 1,554 control subjects. The association between FT3/FT4 ratio and TV among HT patients was then analyzed. We observed a change in laboratory parameters among 13 patients in the large goiter group who were prescribed levothyroxine (LT4) for reducing TV. Compared to normal subjects, the moderate and large goiter groups exhibited significantly higher serum FT3 levels, while all HT groups exhibited lower serum FT4 levels. Serum FT3/FT4 ratios showed a positive correlation with TV (r = 0.35, p < 0.01), which was independent of age, sex, body mass index, and TgAb and TSH levels. LT4 treatment lowered serum FT3 levels and FT3/FT4 ratios significantly. Our results indicated that HT patients with increased TV tended to present with high serum FT3, low FT4, and high FT3/FT4 ratios. The elevation of deiodinase activity may be an important factor affecting thyroid hormonal balance in such patients.

The within- and between-laboratory reproducibility and predictive capacity of the in chemico amino acid derivative reactivity assay: Results of validation study implemented in four participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative method that focuses on protein binding as the molecular initiating event for skin sensitization. It is a simple and versatile method that has successfully solved some of the problems of the direct peptide reactivity assay (DPRA). The transferability and within- and between-laboratory reproducibility of ADRA were evaluated and confirmed as part of a validation study conducted at four participating laboratories. The transfer of ADRA technology from the lead laboratory to the four participating laboratories was completed successfully during a two-step training program, after which the skin sensitization potentials of 40 coded chemicals were predicted based on the results of ADRA testing. Within-laboratories reproducibility was 100% (10 of 10), 100% (10 of 10), 100% (7 of 7) and 90% (9 of 10), or an average of 97.3% (36 of 37); between-laboratory reproducibility as calculated on the results of three laboratories at the time was 91.9%. The overall predictive capacity comprised an accuracy of 86.9%, sensitivity of 81.5% and specificity of 98.1%. These results satisfied the targets set by the validation management team for demonstrating transferability, within- and between-laboratory reproducibility, and predictive capacity as well as gave a clear indication that ADRA is easily transferable and sufficiently robust to be used in place of DPRA.

Cause of and countermeasures for oxidation of the cysteine-derived reagent used in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA-N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)-was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu2+ promoted NAC oxidation significantly. When 0.25 µm of EDTA was added in the presence of Cu2+ , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

Quantitative analysis of γH2AX reveals distinct responses in multiple mouse organs after administration of mitomycin C or ethyl methanesulfonate.

Ser139-phosphorylated H2AX (γH2AX) is a functional biomarker of DNA double-strand breaks. However, its conventional detection for in vivo samples relies on immunological methods using anti-γH2AX antibodies, making quantitative analysis difficult. Here, we established an absolute γH2AX quantification in vivo method for multiple organs in mice using liquid chromatography-triple quadrupole tandem mass spectrometry. When applying the method to male Institute of Cancer Research (ICR) mice (8 weeks old), the testes showed the highest γH2AX level (2.3% of total H2AX), followed by the bone marrow (0.51%), stomach (0.28%), kidney (0.20%), spleen (0.20%), liver (0.15%) and lung, which had the lowest overall level (0.10%). After intraperitoneal administration of 2 mg/kg mitomycin C in mice, γH2AX levels increased until 2-4 h, followed by a monotonical decrease to the control level in the bone marrow and spleen, and increased moderately until 24 h, followed by a slight decrease by 48 h in the liver, stomach, lung and kidney. After oral administration of 400 mg/kg ethyl methanesulphonate, γH2AX levels increased until 8 h and then decreased to the control level by 24-48 h in the spleen and kidney, increased until 24 h and then slightly decreased until 48 h in the bone marrow and lung, increased until 8 h and plateaued by 48 h in the liver, and decreased until 8 h and then increased to the control level in the stomach. Both the genotoxic chemicals did not alter γH2AX levels in the testes. These results indicate that our novel method could reveal variation in the γH2AX state in mouse organs and allows monitoring of the in vivo dynamics induced by genotoxic chemicals.

A novel in chemico method to detect skin sensitizers in highly diluted reaction conditions.

The direct peptide reactivity assay (DPRA) is a simple and versatile alternative method for the evaluation of skin sensitization that involves the reaction of test chemicals with two peptides. However, this method requires concentrated solutions of test chemicals, and hydrophobic substances may not dissolve at the concentrations required. Furthermore, hydrophobic test chemicals may precipitate when added to the reaction solution. We previously established a high-sensitivity method, the amino acid derivative reactivity assay (ADRA). This method uses novel cysteine (NAC) and novel lysine derivatives (NAL), which were synthesized by introducing a naphthalene ring to the amine group of cysteine and lysine residues. In this study, we modified the ADRA method by reducing the concentration of the test chemicals 100-fold. We investigated the accuracy of skin sensitization predictions made using the modified method, which was designated the ADRA-dilutional method (ADRA-DM). The predictive accuracy of the ADRA-DM for skin sensitization was 90% for 82 test chemicals which were also evaluated via the ADRA, and the predictive accuracy in the ADRA-DM was higher than that in the ADRA and DPRA. Furthermore, no precipitation of test compounds was observed at the initiation of the ADRA-DM reaction. These results show that the ADRA-DM allowed the use of test chemicals at concentrations two orders of magnitude lower than that possible with the ADRA. In addition, ADRA-DM does not have the restrictions on test compound solubility that were a major problem with the DPRA. Therefore, the ADRA-DM is a versatile and useful method.

Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals.

To develop a testing strategy incorporating the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non-sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h-CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS-based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h-CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water-soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance.