Osteotoxicity of 3-methylcholanthrene in fish

Abstract

Many chemicals produced by human activities end up in the aquatic ecosystem causing adverse developmental and reproductive effects in aquatic organisms. There is evidence that some anthropogenic chemicals disturb bone formation and skeletal development but the lack of suitable in vitro and in vivo systems for testing has hindered the identification of underlying mechanisms of osteotoxicity. Several fish systems - an in vitro cell system to study extracellular matrix mineralization and in vivo systems to evaluate bone formation and skeletogenesis - were combined to collect data on the osteotoxic activity of 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon. Anti-mineralogenic effects, increased incidence of skeletal deformities and reduced bone formation and regeneration were observed in zebrafish upon exposure to 3-MC. Pathway reporter array revealed the role of the aryl hydrocarbon receptor 2 (Ahr2) in the mechanisms underlying 3-MC osteotoxicity in mineralogenic cell lines. Analysis of gene expression in zebrafish larvae confirmed the role of Ahr2 in the signaling of 3-MC toxicity. It also indicated a possible complementary action of the pregnane X receptor (Pxr) in the regulation of genes involved in bone cell activity and differentiation but also in xenobiotic metabolism. Data reported here demonstrated the osteotoxicity of 3-MC but also confirmed the suitability of fish systems to gain insights into the toxic mechanisms of compounds affecting skeletal and bone formation.

Anti-Osteogenic Activity of Cadmium in Zebrafish

“…Data presented in this article-i.e., the reduced growth of the opercular bone, the increased incidence of skeletal deformities, the impaired stiffness of regenerated bone, and the differential expression of bone marker genes-provide clear evidence of an anti-osteogenic or osteotoxic effect of cadmium at doses that can be found in the aquatic environment [31,32]. A decreased operculum growth rate was recently evidenced in zebrafish larvae exposed to cobalt chloride [33] and 3-methylcholanthrene [30] through mechanisms still poorly understood but possibly related to an impairment of osteoblast (bone forming cells) maturation and function. In this regard, cadmium was found to inhibit extracellular matrix mineralization in osteoblast MC3T3-E1 cell cultures [34] but also to decrease the expression marker genes central to osteoblast maturation and function (this study).…”

“…To get insights into the molecular mechanisms underlying cadmium anti-osteogenic effects, a CIGNAL cell-based reporter assay was used to monitor the activity of 45 signaling pathways involved in processes central to cell biology (see manufacturer website for details). Since cellular hosts of fish origin were found not to be suitable for this analysis (mineralogenic fish cells are hard-to-transfect cellular hosts [30]) and because pathway responsive elements are designed according to mammalian sequences, mouse ATDC5 (chondroprogenitor cells), and MC3T3-E1 (osteoblast precursor cells) lines were used to host the reporter constructs. While the activity of various signaling pathways was detected in both cell lines (21 pathways activated in MC3T3-E1 and ATDC5; see Figure S1 in Supplementary Materials), only 6 pathways showed an activity higher than a 2-fold change over the control.…”

“…Since fish skeletal cell lines are hard-to-transfect cellular hosts [30,85,86] and no commercial reporter arrays are available for fish, mineralogenic cell lines ATDC5 (mouse chondroprogenitor cells) and MC3T3-E1 (mouse osteoblast precursor cells) were used to host the reporter constructs of the Cignal 45-pathway reporter array (QIAGEN). Sub-confluent cultures of ATDC5 and MC3T3-E1 (see the European Collection of Cell Cultures (ECACC) for culture conditions) were reverse-transfected with reporter constructs according to manufacturer protocol.…”

Screening of Mineralogenic and Osteogenic Compounds in Zebrafish—Tools to Improve Assay Throughput and Data Accuracy

“…On the contrary, gilthead seabream VSa13 cell line ( [32]; Cellosaurus accession no. CVCL_S952) was successfully used in small-scale screening of mineralogenic extracts from marine green algae [33], halophytes [34], cyanobacteria (J. Rosa unpublished data), or environmental osteotoxic pollutants [35,36]. Typically, compounds are dissolved in culture medium and exposed to the mineralizing cells in multiwell plates (Figure 1A).…”

Inhibitory Effects of 3-Methylcholanthrene Exposure on Porcine Oocyte Maturation

“…For the male reproductive system, dioxin reduces the number and maturity of sperm cells, causing maternalism, which significantly affects reproduction in animals [ 19 ]. Studies have shown that exposure to 3-MC causes damage to cells, tissue, and organs [ 20 , 21 , 22 , 23 , 24 ]. Numerous studies have shown that 3-MC exposure rechanges cell adhesion and induces cell apoptosis [ 25 , 26 , 27 ], affecting the immunity and metabolism of tissue, which in turn leads to the retention of carcinogenic substances in tissues and makes organs cancerous [ 28 , 29 , 30 , 31 ].…”