Micron-size tire tread particles leach organic compounds at higher rates than centimeter-size particles: Compound identification and profile comparison.

Tire tread particles (TTP) are environmentally prevalent microplastics and generate toxic aqueous leachate. We determined the total carbon and nitrogen leachate concentrations and chemical profiles from micron (∼32 µm) and centimeter (∼1 cm) TTP leachate over 12 days. Dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were used to measure the concentration of leached compounds. Nontargeted chemical analysis by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS) was used to compare the chemical profiles of leachates. After leaching for 12 days, DOC was 4.0 times higher in the micron TTP leachate than in the centimeter TTP leachate, and TDN was 2.6 times higher. The total GC×GC/TOF-MS chromatographic feature peak area was 2.9 times greater in the micron TTP leachate than the centimeter TTP leachate, and similarly, the total relative abundance of 54 tentatively identified compounds was 3.3 times greater. We identified frequently measured tire-related chemicals, such as 6PPD, N-cyclohexyl-N'-phenylurea (CPU), and hexa(methoxymethyl)melamine (HMMM), but nearly 50% of detected chemicals were not previously reported in tire literature or lacked toxicity information. Overall, the results demonstrate that smaller TTP have a greater potential to leach chemicals into aquatic systems, but a significant portion of these chemicals are not well-studied and require further risk assessment.

Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes.

The molecular mechanisms by which overloaded cardiac myocytes increase the cell size (hypertrophy) remain unknown. We have previously shown that mechanical loading increased the protein synthesis and the expression of proto-oncogene c-fos mRNA (Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y. Hoh, E., Takaku, F., and Yazaki, Y. (1990) J. Biol. Chem. 265, 3595-3598; Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., Takaku, F., and Yazaki, Y. (1991) J. Biol. Chem. 266, 1265-1268). It has been known that both mitogen-activated protein (MAP) kinase and S6 kinase can be activated by many kinds of growth factors. To clarify whether MAP kinase(s) and S6 kinase(s) are associated with the intracellular signaling of cardiac hypertrophy induced by mechanical loading, we cultured neonatal rat cardiac myocytes in deformable dishes and imposed an in vitro mechanical loading by stretching the adherent myocytes. In this study, we demonstrated that 1) myocyte stretching maximally activated a kinase activity toward myelin basic protein (MBP) at 10 min after stretching, and the kinase activity returned to the control level at 30 min after stretching; 2) kinase assays in MBP-containing gel, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that stretch-induced MBP kinase activity mainly migrated at 42 kDa in the immunoprecipitated fraction of anti-MAP kinase antibody, suggesting that the stretching mainly increased the 42-kDa MAP kinase activity in cardiac myocytes; 3) phosphorylation of MAP kinase was induced after stretching cardiac myocytes; 4) when protein kinase C was depleted by preincubating myocytes with 100 nM 12-O-tetradecanoyl-phorbol-13-acetate for 24 h or 2 nM staurosporine for 30 min, stretch-induced MBP kinase activity was decreased by approximately 60-70% as compared with the kinase activity in myocytes without protein kinase C depletion; 5) although the receptor tyrosine kinases were depleted by preincubating myocytes with 50 microM tyrphostin or 20 microM genistein for 30 min, there was no change in the stretch-induced MBP kinase activity; 6) stretch-induced MBP kinase activity was partially dependent on transsarcolemmal influx of Ca2+; 7) myocyte stretching also increased S6 peptide (RRLSSLRA) kinase activity in the anti-S6 kinase II antibody immunoprecipitates. From these results, we conclude that myocyte stretching increases the activities of MAP kinase and S6 peptide kinase, which may play an important role in the induction of the specific genes and the increase in the protein synthesis.

Platelet-derived growth factor induces c-fms and scavenger receptor genes in vascular smooth muscle cells.

Vascular smooth muscle cells proliferate and transform to foam cells in the process of atherosclerosis. In the present study, we demonstrated that platelet-derived growth factor (PDGF)-BB induced expression of proto-oncogene c-fms in vascular smooth muscle cells, which normally do not express c-fms, isolated from either human umbilical artery or rabbit aorta. No effect of the protein kinase C activator, phorbol ester, was demonstrated on mRNA expression of c-fms. In contrast, the scavenger receptor activity was induced by both PDGF-BB and phorbol ester. These results indicate that two characteristic genes of monocyte-macrophages were induced by PDGF-BB via the different pathways, and suggest that PDGF-BB plays an important role in initiating phenotypic conversion of smooth muscle cells to macrophage-like cells.

Mechanisms of cardiac hypertrophy and injury--possible role of protein kinase C activation.

To examine the molecular mechanisms by which mechanical stimuli induced cardiac hypertrophy and injury, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos. Marked accumulation of c-fos mRNA followed increases in intracellular Na+ and protein kinase C activation. The accumulation of c-fos mRNA by cardiocyte stretching was suppressed by protein kinase C inhibitors but not by stretch channel blockers. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbolesters stumulated the expression of c-fos. We also examined TGF beta expression in the heart. TGF beta is known to be stimulated by protein kinase C activation, and the mRNA level of TGF beta was increased in in vivo heart by pressure overload. Furthermore, collagen synthesis was stimulated by TGF beta in cultured fibroblasts from hearts. These findings suggest that hemodynamic overload may stimulate cardiac hypertrophy and induce cardiac injury (fibrosis) through protein kinase C activation.

Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. Possible role of protein kinase C activation.

To examine the molecular mechanisms by which mechanical stimuli induce cardiac hypertrophy and specific gene expression, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin. Nuclear run-off transcription assay revealed that this increase in c-fos mRNA level by stretching at least partially reflects changes in the transcriptional status. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene indicated that sequences containing a serum response element were required for efficient transcription by stretching and that sequences containing a cAMP/calcium response element might not be involved in the c-fos response to myocyte stretching. The accumulation of c-fos mRNA by stretching was suppressed by protein kinase C inhibitors at the transcriptional level and inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbol esters stimulated the expression of c-fos and skeletal alpha-actin genes. These findings suggest that mechanical stimuli (myocyte stretching) might directly induce cardiac hypertrophy and specific gene expression possibly via protein kinase C activation.

Molecular mechanism of cardiac hypertrophy.

Pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes. To ascertain the molecular mechanism of these events, we examined the expression of cellular oncogenes and the early change in the translational activity of specific cardiac mRNA by two-dimensional gel electrophoresis of in vitro translational products. Pressure overload increased the expression levels of c-fos, c-myc, and c-Ha-ras genes. The relative predominance of 8 species out of over 400 translational products was increased by pressure overload while that of 2 translational products was decreased. We cloned four pressure-overload-responsive cDNA clones by differential dot blot hybridization. The expression pattern of each cDNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocytes stretching stimulated amino acid uptake and expression of the c-fos gene, which was blocked by protein kinase C inhibitors. These results suggest that there are some early responsive genes in cardiac hypertrophy and that mechanical loading directly stimulates gene expression possibly via protein kinase C activation.

Pseudohypertension in the elderly.

Pseudohypertension is a condition in which indirect blood pressure measured by the cuff method overestimates the true intra-arterial blood pressure. Despite the clinical importance of pseudohypertension, reports regarding its prevalence are scarce. We compared direct measurements of brachial arterial blood pressure with those measured indirectly by the cuff method in 59 healthy volunteers aged greater than 65 years, with or without hypertension, in order to investigate the prevalence of pseudohypertension in the elderly. The average systolic blood pressure of 161.5 mmHg obtained by the indirect method was significantly lower than the 169.2 mmHg obtained by the direct method was not significantly different from the 76.9 mmHg obtained by the direct method. Only one volunteer was observed in whom the indirect cuff method overestimated the diastolic blood pressure by greater than 10 mmHg, which was defined as pseudohypertension. Thus, the prevalence of pseudohypertension was only 1.7%. The difference in systolic blood pressure between the two methods was 8.7 mmHg in Osler-positive cases and was not significantly different from the 6.9 mmHg observed in Osler-negative cases. Pulse wave velocity was significantly correlated with the systolic blood pressure (y = 11.4x + 66.1, r = 0.65, P less than 0.05). No correlation was observed between the pulse wave velocity and direct/indirect pressure differences for either systolic or diastolic measurements. These results show that the prevalence of pseudohypertension is very low in a non-selected elderly population and that Osler's maneuver was not related to the pressure difference between the direct and indirect methods.

Stretching cardiac myocytes stimulates protooncogene expression.

Recently cellular protooncogenes have been found to be induced as an early response to pressure overload in cardiac hypertrophy. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocyte stretching stimulated expression of the protooncogene, c-fos, in a stretch length-dependent manner, followed by an increase in amino acid incorporation into proteins. c-fos mRNA levels were enhanced within 15 min by cardiocyte stretching, peaked at 30 min, and declined to undetectable levels by 240 min. In the presence of cycloheximide, a greater increase in c-fos mRNA was seen by stretching. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene including its promoter was also activated by stretching cardiac myocytes. These results suggest that mechanical loading directly regulates gene transcription without the participation of humoral factors in cardiocytes.

[Simultaneous recording of visually evoked potentials (VEP) and registration of simple visual reaction times (RT) in the maximum range of ethanol influence (author's transl)]. / Gleichzeitige Ableitung visuell evozierter Potentiale (VEP) und Registrierung einfacher visueller Reaktionszeiten (RZ) im "Maximalbereich" de Athanolwirkung

The method of simultaneous recording of visual-evoked response (1 occipit-vertex) and simple reaction time to periodic foveal visual stimuli of 1/" per sec was applied to 16 normal individuals. On two successive days the conditions were kept constant except for an independent variable, i.e., ethanol with an average peak of 1.32%; the range of 0.1% below the peak being examined. The amplitude of the component N2-P3 (120-170 msec after the stimuli) was depressed in all individuals. The latencies of N2 and P3 were increased concurrently. The latency of P3, for example, was increased by 10.7% (P less than or equal 0.01) and that of the means of simultaneously recorded reaction time by 14.8% (P less than or equal 0.01). The amount of the increase, however, showed no significant correlation between the two when using Spearman's correlation coefficient and t-test.