Effect of dietary pristane and other saturated mineral oils (MOSH) on autoimmune arthritis in rats

Abstract

Pristane and other adjuvants based on mineral oil saturated hydrocarbons (MOSH) may induce autoimmunity in rodents after intradermal injection; however there is a lack of information on immune effects after oral MOSH exposure. The aim of our study was to determine the impact of dietary exposure to pristane and other MOSH on the development of autoimmune arthritis. Dark Agouti (DA) rats were given feed containing 4000 mg/kg pristane or a broad MOSH mixture in various concentrations (0-4000 mg/kg) for 90 days, or a single intradermal injection of 200 μl pristane (positive control). Arthritis scores, and serum and splenocyte markers previously associated with arthritis development, were determined. All rats injected with pristane displayed arthritis symptoms and higher levels of certain serum markers. None of the rats fed pristane or MOSH developed arthritis symptoms or demonstrated clear changes in any measured arthritis-associated biological markers in serum or splenocytes. The absence of clinical arthritis symptoms or any increase in common arthritis-associated biological markers in sera and spleen following dietary exposure to pristane or a broad MOSH mixture in a sub-chronic rat model of arthritis suggest that dietary MOSH have low capacity to promote development of autoimmunity.

Keywords: Autoimmune arthritis; DA, Dark Agouti; Dark Agouti rat; MOH, mineral oil hydrocarbons; MOSH, mineral oil saturated hydrocarbons; Mineral oil saturated hydrocarbons (MOSH); RF, rheumatoid factor; TLR, toll like receptor; i.d., intradermal.

Fig. 1

Pilot study. (a) Arthritic scores, (b) body weight (gram; g), and serum levels at termination of (c) IgG-RF, (d) IgM-RF, (e) IL-17 (f), IL-1β and (g) TNFα in female DA-rats intradermally injected on day 0 with 200 μl pristane (n = 6) or physiological buffer (controls, n = 6) and monitored twice a week for up to 40 days. The dots represent the value for the individual animals while the lines represent the group median value. The dotted line indicates the lower detection limit for the ELISA-assay. Asterisk (*) denote statistically significant higher levels compared to the control group (p < 0.05, Mann Whitney test).

Fig. 2

Feed consumption and animal weights. DA-rats were exposed to a single intradermal pristane injection (200 μl), feed containing 4000 mg/kg pristane, or feed containing 0 (control), 40, 400 and 4000 mg/kg of a broad MOSH mixture. (a) Feed consumption (gram; g) week two and week 10 for the dietary exposed male (n = 5) and female (n = 5) rats. (b) Body weight curve (gram; g) for individual male (n = 5) and female (n = 5) DA-rats intradermally injected day 0 with 200 μl pristane and weighed weekly for a maximum of 40 days. (c) Group median body weight (g) for male (n = 5) and female (n = 5) DA-rats, weighed weekly for 90 days. Filled symbols = females, open symbols = males. Injection; circular symbols, pristane in feed; diamonds, 0 mg/kg MOSH; downward triangle, 40 mg/kg MOSH; hexagon, 400 mg/kg MOSH; square, 4000 mg/kg MOSH upward triangle. Two-way ANOVA results (treatment and sex as the two factors) are stated in the figures if statistically significant (p < 0.05).

Fig. 3

Arthritis scores. (a) Arthritic scores in individual DA-rats intradermally injected with 200 μl pristane on day 0 and monitored twice a week for up to 40 days. (b) maximum arthritic scores per animal during the 90 days, in female (closed symbols) and male (open symbols) DA-rats exposed to a single intradermal pristane injection (200 μl), feed containing 4000 mg/kg pristane, or feed containing 0 (control), 40, 400 and 4000 mg/kg of a broad MOSH mixture. The symbols represent the value for the individual animals while the lines represent the group median value. Two-way ANOVA results (treatment and sex as the two factors) are stated in the figures if statistically significant (p < 0.05), and statistically significant group differences (p < 0.05) from the post-hoc test are indicated with brackets.

Fig. 4

Rheumatoid factor and TLR expression. (a) IgG rheumatoid factor (IgG-RF) measured in serum collected at termination, (b) TLR 2 (percent) and (c) TLR 2 (mean fluorescence intensity (MFI)) measured in splenocytes, from female (closed symbols, n = 5) and male (open symbols, n = 5) DA-rats exposed to a single intradermal pristane injection (200 μl), feed containing 4000 mg/kg pristane, or feed containing 0 (control), 40, 400 and 4000 mg/kg of a broad MOSH mixture. The symbols represent the value for the individual animals while the lines represent the group median value. Two-way ANOVA results (treatment and sex as the two factors) are stated in the figures if statistically significant (p < 0.05).

Fig. 5

Cytokine secretion from splenocytes. Concentrations of secreted IL-17a in supernatants of (a) unstimulated (b) LPS and (c) ConA stimulated splenocytes, and (d) IL-10 in supernatants of ConA stimulated splenocytes, from female (closed symbols, n = 5) and male (open symbols, n = 5) DA-rats exposed to feed containing 4000 mg/kg pristane, or feed containing 0 (control), 40, 400 and 4000 mg/kg of a broad MOSH mixture. The symbols represent the value for the individual animals while the lines represent the group median value. Two-way ANOVA results (treatment and sex as the two factors) are stated in the figures if statistically significant (p > 0.05).