DEHP exposure impairs human skeletal muscle cell proliferation in primary culture conditions: preliminary study.

The plasticizer di (2-ethylhexyl) phthalate (DEHP) inhibits differentiation, impairs glucose metabolism, and decreases mitochondrial function in murine muscle satellite cells; however, if these effects are translated to human cells is unknown. The goal of this study was to evaluate changes in morphology and proliferation of primary human skeletal muscle cells exposed to DEHP. Rectus abdominis muscle samples were obtained from healthy women undergoing programed cesarean surgery. Skeletal muscle cells were isolated and grown under standard primary culture conditions, generating two independent sample groups of 25 subcultures each. Cells from the first group were exposed to 1 mM DEHP for 13 days and monitored for changes in cell morphology, satellite cell frequency and total cell abundance, while the second group remained untreated (control). Differences between treated and untreated groups were compared using generalized linear mixed models (GLMM). Cell membrane and nuclear envelope boundary alterations, loss of cell volume and presence of stress bodies were observed in DEHP-treated cultures. DEHP-treated cultures also showed a significant reduction in satellite cell frequency compared to controls. Exposure to DEHP reduced human skeletal muscle cell abundance. Statistical differences were found between the GLMM slopes, suggesting that exposure to DEHP reduced growth rate. These results suggest that exposure to DEHP inhibits human skeletal muscle cell proliferation, as evidenced by reduced cell abundance, potentially compromising long-term culture viability. Therefore, DEHP induces human skeletal muscle cell deterioration potentially inducing an inhibitory effect of myogenesis by depleting satellite cells.

"Oxidative stress induced by phthalates in mammals: State of the art and potential biomarkers".

BACKGROUND: Phthalates, plasticizers that are widely used in consumer products including toys, cosmetics, and food containers, have negative effects in liver, kidney, brain, lung and reproductive system of humans and other mammals. OBJECTIVES: To summarize, describe and discuss the available information on the effects of phthalate exposure in mammals, with emphasis on oxidative stress, and to suggest potential biomarkers of the health risks associated with phthalate exposure. METHODS: An assessment of scientific journals was performed using the PRISMA model for systematic reviews. Manuscripts reporting effects of phthalate exposure on mammalian health published in the last decade were selected according to originality, content, and association to health hazards. RESULTS AND DISCUSSION: We identified 25 peer-reviewed articles published between January 1st, 2010 and June 1st, 2021 that fit the aims and selection criteria. Phthalates induce oxidative stress and cell degenerative processes by increasing intracellular reactive species. Antioxidant cytoprotective systems decrease with time of exposure; conversely, oxidative damage markers, including thiobarbituric acid-reactive substances (TBARS), 8-hydroxy-2'-desoxyguanosine (8-OHdG) and malondialdehyde (MDA), increase. Phthalates were associated with endocrine system disfunction, metabolic disorders, infertility, nonviable pregnancy, cell degeneration, growth impairment, tumor development, and cognitive disorders. Phthalates can also aggravate health conditions such as asthma, hepatitis, diabetes, allergies, chronic liver and kidney diseases. Among humans, the more vulnerable subjects to phthalate exposure effects were children and individuals with a prior health condition. CONCLUSION: Chronic exposure to phthalates induces oxidative stress in mammals with concomitant adverse effects in reproductive, respiratory, endocrine, circulatory, and central nervous systems in both in vitro and in vivo trials. Oxidative damage markers and phthalate metabolites levels were the most common biomarkers of phthalate exposure effects. Studies in free-ranging and wild mammals are nil. Further studies on the pathways that lead to metabolic disruption are needed to identify potential treatments against phthalate-induced detrimental effects.

Morphometric characteristics of human skeletal muscle cells in primary culture / Características morfométricas de células musculares esqueléticas humanas en cultivo primario

SUMMARY: The study of cell morphology has contributed to the innovation of clinical techniques and biomedical research. Primary cell culture techniques are well standardized; however, knowledge about morphometric parameters under cell culture conditions is scarce. Variations in morphology can affect cell physiology and responses. The aim of this study was to use morphometric tools to describe the growth and development of skeletal muscle cells under standard cell culture conditions. A photographic database was generated, and morphometric data was obtained for nine cell characteristics (n = 559 cells). Four muscular cell shapes (spherical, irregular outline, triangular and spindle/fusiform) were characterized with wide ranges in variation. The maximum cell length (110-262 µm), width (35-66 µm), area (2,642 - 9,480 µm2), projection lengths (45 - 127 µm), and nucleus diameter (28 ± 11 µm) were obtained by day 23 of culture. A single centrally positioned nucleus was observed in each cell; nucleoli diameter (5 ± 2 µm) and number (1 - 5) varied. In general, cyclic changes in cell sizes were identified during culture, whereas cell length, width, and area increased in spurts. These results suggest that morphometric parameters can be used to monitor skeletal muscle cell development under standard culture conditions.

RESUMEN: A partir de células madre musculares, surgen los mioblastos que se dividen y fusionan entre sí para formar a los miocitos. Estas células ya diferenciadas son precursoras de miocitos que maduran en fibras musculares y posteriormente forman los músculos. La implementación de cultivos celulares de mioblastos ha permitido obtener conocimiento detallado del tejido muscular. Particularmente, algunas de las aportaciones morfológicas fueron el punto de partida de técnicas clínicas, terapias o investigaciones biomédicas. Sin embargo, los estudios morfométricos en condiciones de cultivo celular son escasos. Por lo cual, realizamos seguimientos fotográficos a cultivos desarrollados bajo condiciones estándar, registramos datos para nueve características celulares y aplicamos técnicas morfométricas para analizar estas células (n = 559). Se caracterizaron cuatro formas celulares adoptadas por los mioblastos (esférica, irregular, triangular y huso) y se registraron intervalos amplios de variación en los caracteres. Hacia el día 23 de cultivo se presentaron los valores máximos en la longitud (110-262 µm), el ancho (35-66 µm) y el área celular (2,642-9,480 µm2), así como en el tamaño máximo de las proyecciones celulares (45-127 µm) y el diámetro del núcleo (28±11 µm). El núcleo se observó como único y en posición central; los nucleolos variaron poco en diámetro (5±2 µm), aunque no en número (1 a 5). En términos generales, se identificaron cambios cíclicos en la talla de las células durante los cultivos, esto es, períodos intercalados de incremento y decremento en el largo, ancho y área celular. Debido a que estas características reflejaron los cambios generales sufridos por los mioblastos durante el cultivo, se proponen para monitorear sus etapas de desarrollo en cultivo.

Morphological alterations in human skeletal muscle cells exposed to di-(2-Ethylhexyl) Phthalate (dehp) in primary cell culture conditions / Alteraciones morfológicas en células musculares esqueléticas humanas expuestas a Ftalato de di-(2-etilhexilo) (dehp) en condiciones de cultivo celular primario

SUMMARY: Di-(2-ethylhexyl) phthalate (DEHP) is among the most common plasticizer additives that humans are in contact with daily. DEHP can be released from plastic and enter the human body, whereby it is metabolized and transformed into oxidative hydrophilic molecules. Clinical follow-ups in patients exposed to this phthalate and investigations in cultures of several cell types have provided information on its effects. For example, it is associated with inhibition of diploid human cell development and morphological changes in cultured germ cells. Although skeletal muscle represents around 50 % of the human body mass, knowledge about the effects of DEHP on this tissue is poor. Cultured skeletal muscle cells were exposed to DEHP (1 mM) for 13 days with the aim of exploring and evaluating some of the potential morphological effects. Three culture development parameters and nine cell characteristics were monitored during the bioassay. At 13 days, growth area, cell viability, and concentration of total proteins were lower in DEHP exposed than in control cells. Cell width and area, as well as the diameter of the nucleus and nucleolus, were greater in exposed cells than in control cells. These are interpreted as signs of cytotoxicity and suggest potential adverse effects on the development of skeletal muscle cells from DEHP exposure, as reported for other cell types.

RESUMEN: Diariamente los seres humanos tenemos contacto con aditivos plastificantes, el di-(2-etilhexil) ftalato (DEHP) se encuentra entre los más comunes. El DEHP puede liberarse del plástico e ingresar al cuerpo humano, donde es metabolizado y transformando en moléculas hidrofílicas oxidativas. Seguimientos en pacientes expuestos a este ftalato e investigaciones en cultivos de varios tipos celulares han aportado información sobre sus efectos. El DEHP es asociado con la inhibición del desarrollo de células humanas diploides y cambios morfológicos en células germinales en cultivo. Sin embargo, aún es poco lo que se sabe sobre los efectos en el músculo esquelético, a pesar de que este tejido representa alrededor del 50 % de la masa corporal del humano. Para explorar y evaluar algunos efectos morfológicos en células de músculo esquelético, cultivos primarios fueron expuestos a DEHP (1 mM) durante 13 días. Se dio seguimiento a tres parámetros de desarrollo del cultivo y nueve características celulares. Al término de 13 días de exposición, los valores del área de crecimiento, viabilidad celular y concentración de proteínas totales fueron inferiores con respecto a los cultivos control. Se observaron cambios morfométricos en las células expuestas. Particularmente, el ancho y área celular, así como los diámetros del núcleo y nucleolos, fueron mayores a los registros en las células control. Estos resultados se interpretan como signos de citotoxicidad y sugieren efectos potencialmente adversos en el desarrollo de las células del músculo esquelético ante una exposición al DEHP, como se ha registrado para otros tipos celulares.

The Pacific harbor seal gut microbiota in Mexico: Its relationship with diet and functional inferences.

Diet is a primary driver of the composition of gut microbiota and is considered one of the main routes of microbial colonization. Prey identification is fundamental for correlating the diet with the presence of particular microbial groups. The present study examined how diet influenced the composition and function of the gut microbiota of the Pacific harbor seal (Phoca vitulina richardii) in order to better understand the role of prey consumption in shaping its microbiota. This species is a good indicator of the quality of the local environment due to both its foraging and haul-out site fidelity. DNA was extracted from 20 fecal samples collected from five harbor seal colonies located in Baja California, Mexico. The V4 region of 16S rRNA gene was amplified and sequenced using the Illumina technology. Results showed that the gut microbiota of the harbor seals was dominated by the phyla Firmicutes (37%), Bacteroidetes (26%) and Fusobacteria (26%) and revealed significant differences in its composition among the colonies. Funtional analysis using the PICRUSt software suggests a high number of pathways involved in the basal metabolism, such as those for carbohydrates (22%) and amino acids (20%), and those related to the degradation of persistent environmental pollutants. In addition, a DNA metabarcoding analysis of the same samples, via the amplification and sequencing of the mtRNA 16S and rRNA 18S genes, was used to identify the prey consumed by harbor seals revealing the consumption of prey with mainly demersal habits. Functional redundancy in the seal gut microbiota was observed, irrespective of diet or location. Our results indicate that the frequency of occurrence of specific prey in the harbor seal diet plays an important role in shaping the composition of the gut microbiota of harbor seals by influencing the relative abundance of specific groups of gut microorganisms. A significant relationship was found among diet, gut microbiota composition and OTUs assigned to a particular metabolic pathway.