Accumulation of methylmercury or polychlorinated biphenyls in in vitro models of rat neuronal tissue.

In vivo exposure levels for neurotoxicants are often reported in parts per million (ppm) concentration in tissue, whereas exposure levels in experiments utilizing in vitro models are most commonly reported in micromolar (muM) concentration in the exposure solution. The present experiments sought to determine whether or not in vitro solution concentration was an appropriate dose-metric for comparison to in vivo tissue levels for lipophilic compounds. To do so, the accumulation of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (A1254) or methylmercury (MeHg) was examined in three commonly utilized in vitro neuronal tissue models: nerve growth factor differentiated pheochromocytoma (PC12) cells, primary cultures of rat neocortical cells, and adult rat hippocampal slices. Tissues were exposed to A1254 (0.65 ppm) or to MeHg (0.0033-0.33 ppm) in serum-free media for 1 or 24 h. Total PCB or mercury accumulation was measured by dual column gas chromatography with electron capture detection or by cold vapor atomic absorption, respectively. PC12 cells accumulated 66.7 and 103.8 ppm PCBs after 1 and 24 h exposure to A1254. Neocortical neurons also accumulated significant concentrations of PCBs, but less so than PC12 cells. After 1 h exposure to 0.65 ppm A1254, slices contained 3.46 and 0.81 ppm PCBs when exposed in a static and perfused system, respectively. After 1 h exposure to 0.0033, 0.033, and 0.33 ppm MeHg, PC12 cells contained 0.3, 2.2, and 17.7 ppm mercury, respectively; after 24 h, PC12 cells contained 0.4, 2.8, and 21.9 ppm. Hippocampal slices accumulated 1.7 and 4.8 ppm mercury after 1 and 3 h exposure to 0.33 ppm MeHg. For comparison, mercury accumulation in rat fetal and pup brain tissue after maternal exposure [0, 0.1, 1.0, or 2.0 mg/kg/day MeHg from gestational day (GD) 6-15] ranged from 0.05 to 7.89 ppm in 0.1 mg/kg dose animals on postnatal day 10 and 2.0 mg/kg dose animals on GD16, respectively. These results demonstrate that accumulation of PCBs and MeHg in vitro is tissue-, time-, and concentration-dependent and indicates that tissue levels rather than exposure concentrations are a more appropriate metric for comparison of in vitro to in vivo effects.

Requirement for G proteins in insulin-like growth factor-I-induced growth of prostate cells.

Elevated levels of IGF-I in the circulation are associated with increased risk for the development of prostate cancer in men, and transgenic expression of human IGF-I in mouse epithelial prostate cells results in spontaneous prostate tumorigenesis. Little, however, is known about the mechanisms involved in the IGF-I-regulated growth of prostate cells. Here, we have demonstrated that treatment with IGF-I induces the activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and the growth of human prostate cells. Stimulation with IGF-I also promoted the tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Signal relay from IGF-I to ERK requires heterotrimeric G proteins and EGFR; inhibition of Gi/o protein activation by pertussis toxin, or EGFR by tyrphostin AG1478 obliterated the ability of IGF-I to promote ERK activation. Further, treatment with pertussis toxin inhibited the IGF-I-mediated prostate cell growth. These data demonstrated the requirement of heterotrimeric G proteins in IGF-I-regulated prostate cell growth and suggest the potential utility of the G proteins as effective drug targets to combat this common cancer.

Abasic sites induce triplet-repeat expansion during DNA replication in vitro.

The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.

Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro.

The human genome contains many simple tandem repeats that are widely dispersed and highly polymorphic. At least one group of simple tandem repeats, the DNA trinucleotide repeats, can dramaticallyexpand in size during transmission from one generation to the next to cause disease by a process known as dynamic mutation. We investigated the ability of trinucleotide repeats AAT and CAG to expand in size during DNA replication using a minimal in vitro system composed of the repeat tract, with and without unique flanking sequences, and DNA polymerase. Varying Mg2+concentration and temperature gave dramatic expansions of repeat size during DNA replication in vitro. Expansions of up to 1000-fold were observed. Mismatches partially stabilized the repeat tracts against expansion. Expansions were only detected when the primer was complementary to the repeat tract rather than the flanking sequence. The results imply that cellular environment and whether the growing strand contains a nick or gap are important factors for the expansion process in vivo.