Effect of 4-Methylbenzylidene Camphor (4-MBC)

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The two substances 4-Methylbenzylidene camphor (4-MBC) and 3-Benzylidene camphor (3-BC) are estrogenic active and lipophilic. These materials find use as sun-screens or product protections in cosmetics (Schlumpf et al. , 2004). Moreover, they are used as ingredients in washing agents for textiles to protect them from damage caused by the sun. UV-Filters can reach mammals through skin or food and can unfold their effects in certain places, including an increase in the weight of the uterus in immature rats (Schlumpf et al.

, 2004). Since the hippocampus contains cells which have estrogen receptors (ERs), the effect of UV filters is of particular importance. The UV filters 4-MBC and 3-BC bind with ER transcription factors, which then attach to certain places of the gene and cause the synthesis of mRNA. These mRNAs are used to synthesize proteins for mitosis (cell proliferation) (Schlumpf et al. , 2004). A similar effect was observed in connection with the proliferating effect of 4-MBC on MCF-7 (mammacarcinoma cells) (Schlumpf et al. , 2001).

Under these circumstances, one assumes that hippocampus cells, especially in the subgranular layer (SGL), will react with cell proliferation to these UV-Filters. The current study aims to provide proof for such proliferating effects in the hippocampus. 2. 1 The Hippocampus Among humans, the brain structure that is directly involved with memory is the hippocampus. The latter is a part of the temporal lobe (The Anatomy Project, 1997). The following is an illustration of this critical structure:

Figure 1. The hippocampus. Source: The Anatomy Project. (1997). Parthenon Publishing Group, p. 67. The hippocampus is involved in the creation of novel memories. For instance, it has been implicated as a critical structure affected by Alzheimer’s disease. Logically, individuals who are afflicted with this disease have faltering memories. Moreover, this structure has also been linked with chronic mental disorders such as depression and schizophrenia; in fact, patients who have been clinically diagnosed with these disorders have smaller hippocampal structures (Woolley, 1999).

Finally, this structure is also strongly influenced by the hormone estrogen, which transcends the latter’s role in reproductive mechanisms (Woolley, 1999). Figure 2. Hippocampus nerve cells: branches. Source: Woolley, 1999, p. 352. Figure 3. Hippocampus nerve cells: Effect of estrogen exposure. Source: Woolley, 1999, p. 353. Figure 2 above illustrates synapses where linkage with other cells transpire. In contrast, Figure 3 depicts the structure with estrogen exposure. Notably, estrogen enhances the density of synapses and allows a greater number of inter-cell connections.

This is specially true for the hippocampus (Woolley, 1999). Figure 4. The dentate gyrus. Source: The Anatomy Project (1997). Parthenon Publishing Group. The hippocampus is made up of complicated folds of the dentate gyrus (Structure 1) and the cornu ammonis (Structure 2). The cornu ammonis is divided in the structures CA1-CA4. The 4 cortices are connected beneath with the subiculum (Structure 3). The latter has 6 layers before it meets with Structure 4, which is the parahippocampal gyrus. Other structures include the lateral genticulate body (5), inferior horn of the lateral ventricle (6) and the fimbria of fornix (7).

The active centers in the dentate gyrus have been investigated by Eriksson et al. (1998) and injected a marker bromodeoxyuridine (BrdU) into cancer patients, with the marker being taken up only by cells who were creating new DNA. The granule cell layer of the dentate gyrus is depicted in the following figure, and it has been identified as the area where DNA creation transpires. Figure 4. Dentate GCL Source: Schacter, 1996, p. 75. The same observations of neurogenesis have been noted with the stain among the hippocampa of mice.

Similar patterns have also been yielded, with the marker appearing at the edge of the granule cell layer (Schacter, 1996). 2. 2 UV-Filters UV-Filters are gradually becoming popular as a research topic, because of their role in the prevention of skin cancer. Moreover, they are used as an ingredient in cosmetic products, in lieu of its characteristics that allow for light stability (Schlumpf et al. , 2001). In their study, Schlumpf et al. (2001) has investigated UVA and UVB screens which have been frequently used. They have examined the estrogenicity of these screens, both in vitro and in vivo.

In their findings, they state that “ in MCF-7 breast cancer cells, five out of six chemicals, that is, benzophenone-3 (Bp-3) , homosalate (HMS) , 4-methyl-benzylidene camphor (4-MBC) , octyl-methoxycinnamate (OMC) , and octyl-dimethyl-PABA (OD-PABA) , increased cell proliferation with median effective concentrations (EC50) values between 1. 56 and 3. 73 µM, whereas butyl-methoxydibenzoylmethane (B-MDM) was inactive“ (Schlumpf et al. , 2001). In addition, there has also been proof on estrogenicity based on 1) the pS2 proteins inducted in MCF-7 cells, and 2) the inhibition of the proliferation of 4-MBC by ICI 182, 780.

The latter is an estrogen antagonist. The assay of rats who have been administered the screens through feed suggest uterine weight increase, dependent on the dose of 4-MBC, OMC, and to a certain extent, Bp-3. These indicate that UV screens must be empirically tested for their effects on endocrine effects (Schlumpf et al. , 2001). In a separate study by Schlumpf et al. (2004), UV screens have again been investigated for endocrine activity. It has been found that in vitro, 8 out of the 9 chemicals examined do have estrogenic MCF-7 cells, while 2 out of 9 have exhibited antiandrogenic effects (MDA-kb2 cells).

The following are the screens which have caused an increased in uterine weight in rats: benzophenone (Bp)-1, Bp-2, Bp-3, 3-benzylidene camphor (3-BC), 4-methylbenzylidene camphor (4-MBC), and octyl-methoxycinnamate (OMC). The effects of 4-MBC (0. 7–47 mg/kg body weight/day) and 3-BC (0. 24–7 mg/kg) administered in feed of rats were examined as regards their effects on postnatal survival and thymus weight when given at different dosages. Among the effects were a delay in reaching puberty among male subjects and repercussions on the development of reproductive organs among offspring.

In particular, the weights of adult male and female rat offspring caused an increase in thyroid weight with increased 4-MBC dosage. There were also effects noted at the histological level, specifically the mRNA levels in the prostate, uterine, and brain (Schlumpf et al. , 2004). 2. 3 Estrogen Since the two UV- Filters are estrgonic active, it is worth discussing this hormone. There are three primary types of estrogens that exist among female organisms. These are estriol, estradiol and estrone (see Figures 5a-5c). Enzymatic activity regulates the production of these estrogens from androgens.

Figure 5a. Estriol: Chemical structure. Source: Fang et al. , 2001, p. 284. Figure 5b. Estradiol: Chemical structure. Source: Fang et al. , 2001, p. 284. Figure 5c. Estrone: Chemical structure. Source: Fang et al. , 2001, p. 285. Estrogens may be found to exist in both male and female organisms, and yet they normally are present at more notable levels among females who are mature in terms of capability to reproduce. The hormone regulates the development of secondary female traits such as breast development, growth of the endometrium, and control of menstruation.

On the other hand, the hormone regulates the maturation of sperm and maintains the sex drive among male organisms (Nussey & Whitehead, 2001). 2. 4 Apoptosis The sustenance of organisms relies strongly on complicated interactions between the cells of the organism and its individual cellular components. Apoptosis, in turn, pertain to programmed death of the cell, which is necessary in the functional development at the histological level (Meier, Finch & Evan, 2000). For instance, in brain development, neurons are later designed to undergo apoptosis once the adult brain has been developed (Zuzarte-Luis & Hurle, 2002).

There has to be an equilibrium achieved in the mechanisms of apoptosis: excessive cell death can cause neurodegenerative disorders or ischemic illnesses. On the other hand, lack of apoptosis can lead to cancer and certain viral diseases (Fadeel Orrenius & Zhivotovsky, 1999). Cells that are undergoing apoptosis may be determined through modifications in its morphology: shrinking, deformation, and losing contact with nearby cells. There is condensation of chromatic and fragmentation of the cell into compact membrane-enclosed bodies. The latter have cytosol, condensed chromatic as well as organelles (Van Chructen & Van Den Broeck, 2002).

Figure 6. Apoptotic versus necrotic morphology. Source:Van Cruchten and Van Den Broeck (2002), p. 220. 3. Material and Methods 3. 1. Chemicals The chemicals which have been used in the current experiment were 4- Methylbenzylidene camphor and 3- Benzylidene camphor. The former has been delivered from Merck (Darmstadt, Germany) and the latter has been purchased from Induchem AG (Volketswil, Switzerland). 3. 2. Animals A total of 18 adult male Long Evans rats have been used in the study, and all laboratory animals were bred in the University of Zurich (Switzerland) animal facilities.

The 18 laboratory animals were randomly assigned to three groups: 1) Untreated control group (6 males); 2) 4-MBC administered with 7 mg/kg per bodyweight intake (6 males); and 3) 3-BC intake 2. 4 mg/kg per bodyweight (6 males). The chemicals 4-MBC and 3-BC were dissolved in soya oil and admixed in chow 3340 (Provimi Kliba AG, Kaiseraugust, Switzerland). The admixed concentrations for 4-MBC have been 100mg/kg chow and for 3-BC 34 mg/kg chow. The average daily intake for the 4-MBC group was 7 mg/kg bodyweight and for the 3-BC group 2. 4 mg/kg bodyweight.

The animals were exposed to food and water 2 weeks long and were held under controlled conditions (lights on 7. 00- 19. 00, 22? 1? C). After the two weeks, the laboratory animals were narcotized and perfused. The anesthetized rats were perfused with phosphate buffered saline (PBS) and then perfused with 4% parafor- maldehyde in 0. 1% phosphate buffer for the postfixation. Afterwards the brains were removed and postfixed for 4 h. Then the brains were cyroprotected in 30% sucrose solution. The brains were then cut in 40-? m parasagittal sections from the right hemisphere and stored in a cryoprotective solution.

3. 3. Immunohistochemistry In the university laboratory the sections were preincubated in 2 % NGS (Normal goat serum, Vectostatin from Vector labs) in 0. 2% Triton in 0. 1% BSA (Bovine serum albumin) in Tris-Triton for 1h. The incubation of the primary antibody Ki67 (1:2500; NCL Ki67p; Novocastra) was carried out overnight at 4? C. The incubation of the secondary antibody (1:1000; goat anti rabbit; Vectastain Elite ABC Kit from Vector Labs) was followed by avidin biotin complex (Vectastain Elite ABC kit from Vector Labs). Afterwards, the staining was performed with DAB as chromogen.

Until the incubation of the primary antibody the rinses were fulfilled with TBS- Triton, then with TBS alone. 3. 4. Cell Counting Each of the 18 subjects was assigned animal codes. For instance, 3BC indicates that the subject has received the 3BC UV screen; the other two codes are 4MBC for the second UV screen, and C to connote the control group. The total numbers of Ki67 positive cells were determined using a 100x oil objective. Ki67 positive cells are an indicator for cell partition and Ki67 is an indicator for such cell proliferation. Apoptosis has also been determined through the use of a microscope.

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